NZ735659B2 - Plasma kallikrein inhibitors and uses thereof for preventing hereditary angioedema attack - Google Patents

Abstract

Provided herein are plasma kallikrein antibodies binding to active plasma kallikrein and methods of using such antibodies in preventing hereditary angioedema attack or reducing the rate of hereditary angioedema attack. A specific embodiment includes the use of the antibody DX-2930 in the manufacture of a medicament for preventing hereditary angioedema (HAE) attack or reducing the rate of HAE attack in a subject, wherein the antibody comprises a heavy chain CDRs 1-3 defined by SEQ ID Nos 5-7 and light chain CDRs defined by SEQ ID Nos 8-10 wherein the antibody is formulated in a pharmaceutically acceptable carrier wherein the medicament is to be administered at about 300 mg of the antibody every two to four weeks at least two times, and wherein the subject is a patient experiencing at least two HAE attacks per year prior to the administration.

Description

Provided herein are plasma rein antibodies binding to active plasma kallikrein and methods of using such antibodies in preventing hereditary angioedema attack or reducing the rate of hereditary angioedema attack. A specific embodiment includes the use of the dy DX-2930 in the manufacture of a ment for preventing hereditary angioedema (HAE) attack or reducing the rate of HAE attack in a subject, wherein the antibody comprises a heavy chain CDRs 1-3 defined by SEQ ID Nos 5-7 and light chain CDRs defined by SEQ ID Nos 8-10 wherein the antibody is formulated in a pharmaceutically acceptable carrier n the medicament is to be administered at about 300 mg of the antibody every two to four weeks at least two times, and wherein the subject is a patient experiencing at least two HAE attacks per year prior to the administration.

NZ 735659 PLASMA KALLIKREIN TORS AND USES THEREOF FOR PREVENTING HEREDITARYANGIOEDEMAATTACK RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of US. provisional application number 62/140,277, filed March 30, 2015, US. provisional application number 62/214,293, filed September 24, 2015 and US. ional application number ,289, filed March 30, 2015, the content of each of which is herein incorporated by nce in its entirety.

BACKGROUND Plasma kallikrein is a serine protease component of the contact system and a potential drug target for different in?ammatory, cardiovascular, infectious (sepsis) and oncology es (Sainz I.M. et al., Thromb Haemost 98, 77—83, 2007). The contact system is activated by either factor XIIa upon exposure to foreign or negatively charged surfaces or on endothelial cell surfaces by prolylcarboxypeptidases (Sainz I.M. et al., Thromb Haemost 98, 77—83, 2007).

Activation of the plasma kallikrein amplifies intrinsic coagulation via its feedback activation of factor X11 and enhances in?ammation via the production of the proin?ammatory nonapeptide bradykinin. As the primary kininogenase in the circulation, plasma kallikrein is largely responsible for the generation of bradykinin in the vasculature. A genetic deficiency in the C l—inhibitor protein (C l—INH), the major natural inhibitor of plasma kallikrein, leads to hereditary angioedema (HAE). Patients with HAE suffer from acute s of painful edema often precipitated by unknown triggers (Zuraw B.L. et al., N Engl J Med 359, 1027—1036, 2008).

SUMMARY The present disclosure is, in part, based on the results derived from clinical studies showing that doses of 0, an dy binding to the active form of human plasma kallikrein, (e.g., 30 mg, 100 mg, 300 mg or 400 mg stered every two weeks) showed unexpected effectiveness in preventing HAE attack or reducing the rate of HAE attack in human ts. Further, DX—2930 treatment did not show evidence of dose—limiting ty when administered to humans. Overall, the results obtained from the instant study were unexpected since DX-2930 is the first completely ic plasma kallikrein inhibitor that has shown high efficacy in HAE treatment. This demonstrates that plasma rein is central to disease pathogenesis.

Accordingly, in a first aspect, the present invention es the use of an antibody in the manufacture of a medicament for preventing hereditary dema (HAE) attack or reducing the rate of HAE attack in a subject, n the antibody comprises a heavy chain (HC) complementarity determining region (CDR) 1 having the amino acid sequence HYIMM (SEQ ID NO: 5), a HC CDR2 having the amino acid sequence GIYSSGGITVYADSVKG (SEQ ID NO: 6), a HC CDR3 having the amino acid sequence RRIGVPRRDEFDI (SEQ ID NO: 7), a light chain (LC) CDR1 having the amino acid sequence RASQSISSWLA (SEQ ID NO: 8), a LC CDR2 having the amino acid sequence KASTLES (SEQ ID NO: 9), and a LC CDR3 having the amino acid sequence QQYNTYWT (SEQ ID NO: 10), wherein the antibody is formulated in a pharmaceutically acceptable carrier comprising sodium phosphate at a concentration of 30 mM, citric acid, histidine at a concentration of 50 mM, sodium chloride at a concentration of 90 mM, and polysorbate 80 at 0.01%, pH 6.0, wherein the medicament is to be administered of the dy every two to four weeks at least two times, and wherein the subject is a patient experiencing at least two HAE s per year prior to the administration.

One aspect of the present disclosure features a method of preventing HAE attack or reducing the rate of HAE attack (e.g., type I, II, or III HAE), the method comprising administering to a subject in need thereof an antibody binding to the active form of human plasma kallikrein (e.g., DX-2930) in an effective amount (e.g., about 30 mg -400 mg, about 100 mg – 400 mg, about 100 mg - 300 mg, or about 300 mg - 400 mg). In some ments, the antibody is administered every two to four weeks for at least two times. In some embodiments, the dy is administered at 300 mg or 400 mg every 2 weeks to four weeks (e.g., every two weeks or every four weeks).

In any one of the methods described herein, the antibody can be administered by subcutaneous administration. In some embodiments, the subject is a human t experiencing at least two HAE attack per year (e.g., at least one HAE attack within 6 month prior to the first administration, at least 2 HAE attacks within 3 months prior to the first administration, or at least 9 HAE attacks within 3 months prior to the first administration).

The HAE can be type I HAE or type II HAE. For example, the method described herein is for prophylactic treatment of HAE.

The antibody used in any of the methods described herein may be an antibody (e.g., a ength antibody or an antigen-binding fragment) that binds the same epitope as DX-2930 or competes against DX-2930 for binding to active human plasma kallikrein. In some ments, the antibody comprises the same heavy chain and light chain CDRs. In one example, the antibody is DX-2930. Any of the antibody as described herein (e.g., DX-2930) may be formulated in a pharmaceutical ition, which comprises a pharmaceutically acceptable r. In some examples, the pharmaceutical composition comprises sodium phosphate, citric acid, histidine, sodium chloride, and Tween 80. In one example, the antibody (e.g., DX-2930) is formulated in 30 mM sodium phosphate, 8.6 mM citric acid, 50 mM histidine, 90 mM sodium chloride, and 0.01% Tween 80, pH 6.0.

In yet other aspects, the t disclosure features a method of treating HAE (e.g., type I, II, or III), the method comprising administering to a subject in need thereof an antibody binding to the active form of human plasma kallikrein (e.g., DX- 2930) in an effective amount (e. g., 100—400 mg, 100—300 mg, 150 mg or 300 mg), wherein the DX—2930 antibody is administered in a dosage regimen having a loading period (e.g., first administered every week such as for at least one week), a maintenance period (e. g., subsequently administered every two to four weeks), and ally a —on period.

In some embodiments, the antibody is administered to the subject at 100 to 300 mg (e.g., 150 mg or 300 mg) during the loading period. The loading period may be two weeks. The antibody may be administered at day 0, day 7, and day 14 at, e.g., 150 mg or 300 mg.

Alternatively or in addition, the dy is administered to the subject at 100 to 300 mg (e.g., 150 mg or 300 mg) during the maintenance period. The maintenance period may last for 10 weeks. The antibody can be administered at day 28, day 42, day 56, day 70 and day 84.

In any of the methods described herein, the method may further comprise administering to the subject the antibody after the maintenance period once every two to four weeks (e.g., every two weeks or every four weeks). In some examples, the antibody is administered at 100 to 400 mg (e.g., 100 mg to 300 mg, for example, 150 mg or 300 mg).

In some ments of any one of the methods described herein, the dy can be administered by subcutaneous administration. In some embodiments, the subject is a human patient suffering from, suspected of having, or at risk for HAE attack. For example, the method described herein is for prophylactic treatment of HAE. The subject may be a human patient who experienced at least 2 attacks per year (e.g., at least one attack per 4 weeks) prior to the treatment. In some embodiments, the antibody is administered for preventing HAE attack or for reducing the rate of HAE attack.

In some ments, the antibody (e.g., DX-2930) is first administered every week for one, two or three weeks and subsequently administered every two, three or four weeks. In some embodiments, the antibody (e.g., DX—2930) is subsequently administered every two weeks for ten weeks. In some ments, the subject has at least one attack every four weeks prior to the first administration.

The antibody to be used in any of the s described herein may be an antibody that binds the same epitope as 0 or competes against DX—293O for binding to active human plasma kallikrein. In some embodiments, the antibody comprises the same heavy chain and light chain CDRs. In one example, the antibody is 0.

Also within the scope of the present disclosure are (a) pharmaceutical compositions for use in treating HAE (e.g., preventing HAE attack or reducing the rate of HAE attack), the pharmaceutical composition comprising any of the anti— rein antibodies described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered to a subject ing any of the treatment regimens described herein; and (b) use of the ceutical composition for cturing a medicament for the treatment of HAE. Use of the antibodies for the ed purposes could be performed under the treating regimens as described .

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed ption of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS FIGURE 1 shows DX—2930 plasma drug levels following subcutaneous dosing in HAE patients in the phase lb study.

FIGURE 2 shows ?uorogenic activity assay of HAE patient samples from the phase lb study.

FIGURE 3 is a Western blot analysis of SCAT169 plasma from HAE patients from the phase lb study.

FIGURE 4 is a Western blot analysis of citrated plasma from HAE patients from the phase 1b study.

FIGURE 5 is a Western blot analysis of citrated plasma activated ex vivo with FXIIa from HAE patients from the phase 1b study.

FIGURE 6 shows the primary efficacy assessment period for ts in different dosing cohorts. A.: 300 mg cohort. B.: 400 mg cohort. Red bars show the interval assessed for efficacy.

FIGURE 7 shows the reduction in HAE attack rate in patients treated with 300 mg, 400 mg, combined (300 and 400 mg) or placebo. Baseline was defined as historical HAE attacks over last 3 months prior to dosing. The data includes patients with a baseline rate of Z 2 attacks in the last 3 months. Day 8 to 50 attack rates were unadjusted for baseline rates. t reduction in HAE attack rate over placebo and p—value were calculated based upon Mixed Model Repeated Measurements with Analysis of ce (baseline attack rate as covariate) and assuming n distribution.

FIGURE 8 shows the nce of HAE attacks in placebo—treated subjects.

The X-axis shows the study day number.

FIGURE 9 shows the mean DX-2930 concentration and HAE attack nce following 30 mg dosage.

FIGURE 10 shows the mean DX—2930 tration and HAE attack incidence following 100 mg dosage.

FIGURE 11 shows the mean DX—2930 concentration and HAE attack incidence following 300 mg dosage.

FIGURE 12 shows the mean DX—2930 concentration and HAE attack incidence following 400 mg dosage. Excludes one patient who received only one dose (in order to derive the mean pharmacokinetic .

FIGURE 13 shows DX—2930 concentration and HAE attacks in patients with historical attack rate 2 9 attacks/3months. A.: a placebo patient. B.: a patient treated with 300 mg. and C.-F.: patients treated with 400 mg.

FIGURE 14 shows an exemplary dosing regimen comprising a loading period and a maintenance period, which may be followed by an extended ent period or a t period.

DETAILED DESCRIPTION Definitions For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are defined here.

Other terms are defined as they appear in the speci?cation.

The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

The term “antibody” refers to a protein that includes at least one immunoglobulin variable domain ble region) or immunoglobulin variable domain (variable ) sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH or HV), and a light (L) chain le region (abbreviated herein as VL or LV). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen—binding fragments of antibodies (e. g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39)) as well as complete dies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).

Antibodies may be from any , but primate (human and non-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions of ariability, termed ementarity determining regions” (“CDRs”), persed with regions that are more conserved, termed “framework regions” (“FRs”). The extent of the framework region and CDRs have been defined (see, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US. ment of Health and Human Services, NIH Publication No. 91— 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901—917). Kabat definitions are used herein. Each VH and VL is typically ed of three CDRs and four FRs, arranged from amino—terminus to carboxy—terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain such that one or more CDR regions are positioned in a conformation suitable for an antigen binding site. For e, the sequence may include all or part of the amino acid sequence of a naturally—occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other tions. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially cts with plasma kallikrein.

The VH or VL chain of the dy can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light globulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter—connected by, e.g., ide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen.

The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody—dependent cytotoxicity and/or ment—mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and/or LC CDR3. Each of the light chain (LC) and/or heavy chain (HC) CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and/or FR4 of the HC and/or LC. For example, the Fc region can be human. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non—hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a ne nucleic acid. In one embodiment, the framework (FR) residues of a selected Fab can be converted to the acid type of the corresponding residue in the most similar primate germline gene, ally the human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of an globulin variable domain, the constant region, the constant domains (CH1, CH2, CH3, and/or CLl), or the entire dy can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, n and mu constant region genes, as well as the many immunoglobulin variable region genes.

Full—length immunoglobulin “light chains” (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2—terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full—length immunoglobulin “heavy chains” (about 50 KDa or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The length of human HC varies considerably because HC CDR3 varies from about 3 amino—acid residues to over 35 amino—acid residues.

The term en—binding fragment” of a full length antibody refers to one or more fragments of a full—length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments assed within the term “antigen—binding fragment” of a full length antibody and that retain functionality include (i) a Fab fragment, a monovalent nt consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent nt including two Fab fragments linked by a disul?de bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH s of a single arm of an dy, (v) a dAb fragment (Ward et al., (1989) Nature 341:544—546), which consists of a VH domain; and (vi) an isolated complementarity ining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a tic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See, e.g., US. Pat. Nos. 5,260,203, 778, and 4,881,175; Bird et a1. (1988) Science 242:423—426; and Huston et a1. (1988) Proc. Natl. Acad. Sci. USA 9-5883.

Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art. The term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a “monoclonal antibody” or “monoclonal dy composition,” which as used herein refers to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.

Antibodies are “germlined” by reverting one or more non—germline amino acids in framework regions to ponding germline amino acids of the antibody, so long as g properties are substantially retained.

The inhibition constant (Ki) provides a measure of tor potency; it is the concentration of inhibitor required to reduce enzyme activity by half and is not dependent on enzyme or substrate concentrations. The apparent Ki (Ki,app) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of inhibitor (e.g., inhibitory binding protein) on the extent of the reaction (e.g., enzyme activity); ?tting the change in pseudo-first order rate constant as a function of inhibitor tration to the Morrison equation (Equation 1) yields an estimate of the apparent Ki value. The Ki is obtained from the rcept extracted from a linear regression analysis of a plot of Ki,app versus substrate concentration.

(KW, + I + E)— (KW, + I + E) —42 - I - E v 2 v0 — v0 Equation 1 Where v = measured velocity; v0 = velocity in the absence of inhibitor; Ki,app = apparent inhibition constant; I = total inhibitor concentration; and E = total enzyme concentration.

As used herein, “binding affinity” refers to the apparent association nt or KA. The KA is the reciprocal of the dissociation constant (KD). A binding antibody may, for example, have a binding af?nity of at least 105, 106, 107, 108, 109, 1010 and 1011 M-l for a ular target molecule, e.g., plasma kallikrein. Higher affinity binding of a binding antibody to a first target ve to a second target can be ted by a higher KA (or a smaller numerical value KD) for binding the first target than the KA (or numerical value KD) for binding the second target. In such cases, the binding antibody has specificity for the first target (e.g., a n in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, , 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105 fold.

Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel tion, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a ?uorescence assay). Exemplary conditions for evaluating binding affinity are in HBS—P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These ques can be used to measure the concentration of bound and free binding protein as a function of binding protein (or target) tration. The concentration of bound binding protein ([Bound]) is related to the tration of free g n ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation: [Bound] = N - [Free]/((1/KA) + [Free]).

It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of ty, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e. g., 2 fold higher, to obtain a ative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The term “binding antibody” (or “binding protein” used interchangeably herein) refers to an antibody that can interact with a target molecule. This term is used interchangeably with “ligand.” A “plasma kallikrein binding antibody” refers to an antibody that can interact with (e.g., bind) plasma kallikrein, and includes, in particular, dies that preferentially or specifically ct with and/or inhibit plasma kallikrein. An antibody inhibits plasma kallikrein if it causes a decrease in the activity of plasma kallikrein as compared to the activity of plasma kallikrein in the absence of the antibody and under the same ions.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, ne, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, , threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta—branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

It is possible for one or more framework and/or CDR amino acid es of a binding protein to include one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino , insertions, or deletions) relative to a binding protein described herein. A plasma kallikrein binding protein may have ons (e.g., substitutions (e.g., vative substitutions or substitutions of non—essential amino acids), insertions, or deletions) (e.g., at least one, two, three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations) relative to a binding protein described herein, e.g., mutations which do not have a substantial effect on protein on. The ons can be present in framework regions, CDRs, and/or constant s. In some embodiments, the mutations are present in a framework region. In some embodiments, the mutations are present in a CDR. In some ments, the mutations are present in a constant region. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity, can be predicted, e.g., by evaluating whether the mutation is conservative or by the method of Bowie, et al. (1990) Science 247:1306—1310.

An “effectively human” immunoglobulin variable region is an globulin le region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An “effectively human” antibody is an antibody that includes a suf?cient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.

An “epitope” refers to the site on a target compound that is bound by a binding protein (e. g., an antibody such as a Fab or full length antibody). In the case where the target compound is a protein, the site can be ly composed of amino acid components, entirely composed of al modi?cations of amino acids of the protein (e. g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue, yl group, phosphate group, sulfate group, or other molecular feature.

A first binding antibody “binds to the same epitope” as a second binding antibody if the first g antibody binds to the same site on a target compound that the second binding antibody binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group)) with the site that the second binding antibody binds.

A first binding dy “competes for binding” with a second binding antibody if the binding of the first binding antibody to its epitope decreases (e.g., by %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second binding antibody that binds to its epitope. The competition can be direct (e.g., the first binding antibody binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding antibody), or indirect (e.g., the binding of the first binding antibody to its epitope causes a steric change in the target compound that decreases the y of the second binding antibody to bind to its epitope).

Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are med as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or c acid ce for optimal alignment and mologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid ons or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or tide as the corresponding position in the second ce, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, ably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% of the length of the nce sequence. For example, the reference sequence may be the length of the immunoglobulin variable domain sequence.

A “humanized” globulin variable region is an immunoglobulin le region that is ed to include a suf?cient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an genic response in a normal human. Descriptions of “humanized” immunoglobulins include, for example, US. 6,407,213 and US. 5,693,762.

An “isolated” antibody refers to an antibody that is removed from at least 90% of at least one component of a natural sample from which the isolated antibody can be obtained. Antibodies can be “of at least” a certain degree of purity if the s or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight—weight basis.

A “patient,99 63subject” or “host” (these terms are used interchangeably) to be d by the subject method may mean either a human or non—human animal.

The terms “prekallikrein” and “preplasma kallikrein” are used interchangeably herein and refer to the zymogen form of active plasma kallikrein, which is also known as likrein.

As used herein, the term “substantially identical” (or “substantially homologous”) is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or lent (e.g., with a similar side chain, e. g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) similar activities, e.g., a binding activity, a binding preference, or a biological activity. In the case of antibodies, the second dy has the same specificity and has at least 50%, at least %, or at least 10% of the affinity relative to the same antigen.

Sequences similar or homologous (e.g., at least about 85% sequence identity) to the ces disclosed herein are also part of this application. In some embodiments, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, a plasma kallikrein binding antibody can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an antibody described herein. In some embodiments, a plasma kallikrein binding antibody can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LC framework regions (e.g., HC and/or LC FR 1, 2, 3, and/or 4) to an antibody described herein (e.g., DX-2930). In some embodiments, a plasma kallikrein binding antibody can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence ty in the HC and/or LC CDRs (e.g., HC and/or LC CDRl, 2, and/or 3) to an dy described herein (e.g., DX-2930). In some embodiments, a plasma kallikrein binding antibody can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the nt region (e.g., CH1, CH2, CH3, and/or CLl) to an antibody described herein (e.g., DX—2930).

In addition, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

Statistical significance can be determined by any art known method.

Exemplary statistical tests include: the Students T—test, Mann y U non— parametric test, and Wilcoxon non—parametric tical test. Some statistically icant relationships have a P value of less than 0.05 or 0.02. Particular binding proteins may show a difference, e.g., in speci?city or binding that are statistically significant (e.g., P value < 0.05 or 0.02). The terms “induce”, “inhibit39 4‘ , potentiate”, “elevate”, “increase”, ase” or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, may refer to a ence, e.g., a tically significant difference, between the two states.

A “therapeutically ive dosage” ably modulates a measurable parameter, e.g., plasma kallikrein activity, by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to modulate a measurable parameter, e.g., a disease—associated parameter, can be evaluated in an animal model system predictive of efficacy in human disorders and conditions. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.

The term “treating” as used herein refers to the application or administration of a ition including one or more active agents to a subject, who has an allergic disease, a symptom of the allergic disease, or a predisposition toward the allergic disease, with the purpose to cure, heal, alleviate, e, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the e, or the predisposition toward the disease. “Prophylactic treatment,” also known as “preventive treatment,” refers to a treatment that aims at protecting a person from, or ng the risk for a disease to which he or she has been, or may be, exposed.

The term “preventing” a e in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the stration of a drug, such that at least one symptom of the disease is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition. “Preventing” a e may also be ed to as “prophylaxis” or “prophylactic treatment.” A ylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to e the desired prophylactic result. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Plasma kallikrein binding antibodies for use in the methods described herein can be full—length (e.g., an IgG (e.g., an IgGl, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgAl, IgAZ), IgD, and IgE) or can include only an antigen-binding fragment (e.g., a Fab, F(ab’)2 or scFv fragment. The binding antibody can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody. Plasma kallikrein binding antibodies can be recombinant ns such as humanized, CDR grafted, chimeric, nized, or in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences. In one embodiment, the plasma kallikrein binding antibody is a monoclonal antibody.

In one aspect, the disclosure features an antibody (e.g., an isolated antibody) that binds to plasma kallikrein (e.g., human plasma kallikrein and/or murine kallikrein) and es at least one immunoglobulin variable . For example, the antibody includes a heavy chain (HC) globulin variable domain sequence and/or a light chain (LC) globulin variable domain sequence. In one embodiment, the antibody binds to and inhibits plasma kallikrein, e.g., human plasma kallikrein and/or murine kallikrein.

The antibody can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin le domain ce comprises one or more (6.3., 1, 2, or 3) CDRs that are at least 85 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC le domain described herein; (c) the LC immunoglobulin variable domain ce comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described ; ((1) the LC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain described herein (e.g., overall or in framework regions or CDRs); (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variable domain described herein (e.g., overall or in ork regions or CDRs); (f) the antibody binds an epitope bound by an antibody described herein, or competes for binding with an antibody described herein; (g) a primate CDR or primate framework region; (h) the HC immunoglobulin variable domain sequence comprises a CDRl that differs by at least one amino acid but by no more than 2 or 3 amino acids from the CDRl of a HC variable domain described ; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domain described herein; 0) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid but by no more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variable domain described herein; (k) the LC immunoglobulin variable domain sequence comprises a CDRl that differs by at least one amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDRl of a LC variable domain described herein; (1) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, or 4 amino acids from the CDR2 of a LC variable domain described herein; (m) the LC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDR3 of a LC le domain described herein ; (n) the LC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a LC variable domain described herein (e.g., overall or in ork regions or CDR3); and (o) the HC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a HC variable domain described herein (e.g., overall or in framework regions or CDRs).

The plasma rein binding protein may be an isolated antibody (e.g., at least 70, 80, 90, 95, or 99% free of other proteins). In some embodiments, the plasma rein binding antibody, or composition thereof, is ed from antibody cleavage fragments (e.g., DX—2930) that are inactive or partially active (e.g., bind plasma kallikrein with a Ki, app of 5000 nM or greater) ed to the plasma kallikrein binding antibody. For example, the plasma kallikrein binding antibody is at least 70% free of such antibody ge fragments; in other embodiments the binding antibody is at least 80%, at least 90%, at least 95%, at least 99% or even 100% free from antibody cleavage fragments that are inactive or partially active.

The plasma rein binding antibody may additionally inhibit plasma kallikrein, e.g., human plasma kallikrein.

In some embodiments, the plasma kallikrein binding antibody does not bind prekallikrein (e.g., human prekallikrein and/or murine prekallikrein), but binds to the active form of plasma rein (e.g., human plasma kallikrein and/or murine kallikrein).

In certain embodiments, the antibody binds at or near the active site of the catalytic domain of plasma rein, or a fragment thereof, or binds an epitope that overlaps with the active site of plasma rein.

In some aspects, the antibody binds the same epitope or competes for binding with an antibody described herein.

The antibody can bind to plasma kallikrein, e.g., human plasma kallikrein, with a binding affinity of at least 105, 106, 107, 108, 109, 1010 and 1011 M1. In one embodiment, the antibody binds to human plasma kallikrein with a Koff slower than 1 X 103, 5 X 10¢l S4, or 1 X 10'4 s‘l. In one embodiment, the dy binds to human plasma kallikrein with a Kon faster than 1 X 102, l X 103, or 5 X 103 M'ls'l. In one embodiment, the antibody binds to plasma kallikrein, but does not bind to tissue kallikrein and/or plasma likrein (e.g., the antibody binds to tissue kallikrein and/or plasma prekallikrein less effectively (e.g., 5-, 10-, 50-, 100-, or 1000-fold less or not at all, e. g., as compared to a negative control) than it binds to plasma kallikrein, In one embodiment, the antibody inhibits human plasma kallikrein activity, e.g., with a Ki of less than 105, 106, 10”, 10*, 109, and 10‘10 M. The antibody can have, for example, an IC50 of less than 100 nM, 10 nM, l, 0.5, or 0.2 nM. For example, the antibody may modulate plasma kallikrein activity, as well as the production of Factor XIIa (e.g. , from Factor XII) andlor bradykinin (e.g., from high— molecular—weight kininogen (HMWK)). The antibody may inhibit plasma kallikrein activity, and/or the production of Factor XIIa (e.g., from Factor XII) and/or bradykinin (e.g., from high—molecular—weight gen (HMWK)). The affinity of the antibody for human plasma kallikrein can be characterized by a KD of less than 100 nm, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. In one embodiment, the antibody inhibits plasma kallikrein, but does not inhibit tissue kallikrein (e. g., the dy inhibits tissue kallikrein less effectively (e. g., 5—, 10—, 50— or 1000—fold less or not at all, e.g., as compared to a negative control) than it , 100—, inhibits plasma kallikrein.

In some embodiments, the antibody has an apparent inhibition nt (KLapp) of less than 1000, 500, 100, 5, l, 0.5 or 0.2 nM.

Plasma rein binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e. g., IgG or Fab).

In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of ent polypeptide chains. For example, the antibody is an IgG, e. g., IgGl, IgG2, IgG3, or IgG4. The antibody can be a soluble Fab. In other implementations the dy es a Fab2', scFv, minibody, scszch fusion, FabzzHSA fusion, HSA::Fab fusion, FabzzHSAzzFab fusion, or other molecule that comprises the antigen ing site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LszHSA + l, HSA::LC + VH::CHl, or other appropriate construction.

In one embodiment, the antibody is a human or humanized antibody or is non— immunogenic in a human. For example, the antibody includes one or more human dy framework regions, e. g., all human framework regions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to human framework regions. In one embodiment, the antibody es a human Fc , or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc .

In one embodiment, the antibody is a primate or primatized antibody or is non— immunogenic in a human. For example, the antibody includes one or more primate antibody framework regions, e.g., all primate framework regions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to primate framework regions. In one ment, the antibody includes a primate Fc domain, or an Fc domain that is at least 95 or 99% identical to a e Fc , 96, 97, 98, domain. “Primate” includes humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas (Gorilla a), gibons, monkeys, lemurs, aye—ayes (Daubentonia madagascariensis), and tarsiers.

In some ments, the affinity of the primate antibody for human plasma kallikrein is characterized by a KD of less than 1000, 500, 100, 10, 5, l, 0.5 nM, e.g., less than 10 nM, less than 1 nM, or less than 0.5 nM.

In certain embodiments, the antibody includes no sequences from mice or rabbits (e. g., is not a murine or rabbit antibody).

In some embodiments, the antibody used in the methods described herein may be DX-2930 as bed herein or a functional variant thereof, or an antibody that binds the same epitope as DX—2930 or competes against DX—2930 for binding to active plasma kallikrein.

In one example, a functional variant of DX—2930 comprises the same complementary ining regions (CDRs) as 0, as determined by the same . In another example, the functional variants of DX—2930 may contain one or more mutations (e.g., conservative substitutions) in the FRs of either the VH or the VL as compared to those in the VH and VL of DX—2930. Preferably, such mutations do not occur at residues which are predicted to interact with one or more of the CDRs, which can be determined by routine logy. In other embodiments, the functional variants described herein n one or more mutations (e.g., l, 2, or 3) within one or more of the CDR regions of DX—2930. Preferably, such onal variants retain the same regions/residues responsible for antigen—binding as the parent. In yet other embodiments, a functional variant of DX-2930 may comprise a VH chain that comprises an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to that of the VH of DX—2930 and/or a VL chain that has an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to that of the VL of 0. These variants are capable of binding to the active form of plasma kallikrein and preferably do not bind to prekallikrein.

The nt 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 in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873—77, 1993.

Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, er al. J. Mol. Biol. 215:403—10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein les of interest. Where gaps exist n two sequences, Gapped BLAST can be utilized as described in Altschul er al., Nucleic Acids Res. 25(17):3389—3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and ) can be used.

In some embodiments, the antibody used in the methods and compositions described herein may be the DX—2930 antibody. The heavy and light chain full and variable sequences for DX-2930 are ed below, with signal sequences in italics.

The CDRs are boldfaced and underlined (based on the Kabat numbering ).

DX—293O Heavy Chain Amino Acid Sequence (451 amino acids, 49439.02 Da) MGWSCILFLVATATGAHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMM GKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1) DX—2930 Light Chain Amino Acid Sequence (213 amino acids, 23419.08 Da) MGWSCILFLVATATGAHSDIQMTQSPSTLSASVGDRVTITCRASSQSISSWLAWY QQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCm YNTYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) DX—293O Heavy Chain Variable Domain Amino Acid Sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSQ TVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIG VPRRDEFDIWGQGTMVTVSS (SEQ ID NO: 3) DX—2930 Light Chain Variable Domain Amino Acid Sequence DIQMTQSPSTLSASVGDRVTITCRASSQSISSWLAWYQQKPGKAPKLLIYKAST SRFSGSGSGTEFTLTISSLQPDDFATYYCS 2gzYNTYWTFGQGTKVEIK (SEQ ID NO: 4) Table 1. CDRs for DX-2930.

HeavychainCDR2 GIYSSGGITVYADSVKG (SEQ ID NO: 6) QQYNTYWT (SEQ ID NO: 10) Antibody Preparation An antibody as described herein (e.g., DX-2930) can be made by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York and Greenfield, (2013) dies: A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press.

The sequence encoding the antibody of interest, e.g., DX—2930, may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to “humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in al trials and treatments in humans. It may be desirable to genetically manipulate the dy sequence to obtain r ty to the target antigen and greater efficacy in inhibiting the ty of PKal. It will be nt to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other embodiments, fully human dies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. enic animals that are designed to produce a more desirable (e. g., fully human antibodies) or more robust immune response may also be used for generation of zed or human antibodies. Examples of such logy are XenomouseRTM from Amgen, Inc. (Fremont, Calif.) and HuMAb—MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, NJ.) In another alternative, antibodies may be made recombinantly by phage y or yeast technology. See, for example, US. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Antigen—binding nts of an intact antibody (full—length antibody) can be prepared via routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of an dy molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.

Genetically engineered dies, such as humanized antibodies, chimeric antibodies, single—chain dies, and bi—specific dies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated or synthesized. The DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the inant host cells. See, e. g., PCT ation No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 8126851, or by ntly joining to the immunoglobulin coding sequence all or part of the coding sequence for a non— immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies; can be prepared that have the binding icity of a target antigen.

Techniques ped for the production of “chimeric antibodies” are well known in the art. See, e.g., Morrison et a1. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 3 14:452.

Methods for constructing humanized antibodies are also well known in the art.

See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029—10033 (1989). In one example, variable regions of VH and VL of a parent non—human dy are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid ces that are homologous to those of the parent non—human antibody are identified from any antibody gene database using the parent VH and VL ces as search queries. Human VH and VL acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non—human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are ted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology by g a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a ?exible linker is incorporated between the two variable regions. Alternatively, ques described for the production of single chain antibodies (US. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv library and scFv clones specific to a PKal can be identified from the library following routine ures. ve clones can be subjected to further screening to identify those that inhibits PKal activity.

Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells (see e.g., Nadkarni, A. et al., 2007 Protein Expr Purif 52(1):2l9—29). For example, if the Fab is encoded by sequences in a phage display vector that es a suppressible stop codon between the y entity and a iophage protein (or fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene 111 protein and is secreted into the periplasm and/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment, the dies (e. g., scFv’s) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al., 2001, J. Immunol. Methods. 251: 123—35; Schoonooghe S. et al., 2009 BMC Biotechnol. 9:70; Abdel-Salam, HA. et al., 2001 Appl Microbiol Biotechnol 56(1— 2):157-64; Takahashi K. et al., 2000 Biosci Biotechnol Biochem 64(10):2138-44; Edqvist, J. et al., 1991 J Biotechnol 291-300), Hanseula, or Saccharomyces.

One of skill in the art can optimize antibody production in yeast by zing, for example, oxygen conditions (see e.g., Baumann K., et al. 2010 BMC Syst. Biol. 4:141), osmolarity (see e.g., its, M. et al., 2010 BMC Genomics 11:207), temperature (see e.g., Dragosits, M. et al., 2009 J Proteome Res. 8(3):l380—92), fermentation conditions (see e.g., Ning, D. et al. 2005 J. Biochem. and Mol. Biol. 38(3): 294—299), strain of yeast (see e.g., Kozyr, AV et a1. 2004 M01 Biol (Mosk) 38(6):1067—75; Horwitz, AH. et al., 1988 Proc Natl Acad Sci U S A 85(22):8678—82; Bowdish, K. et a1. 1991 J Biol Chem ): 1 1901-8), overexpression of proteins to e antibody production (see e.g., Gasser, B. et al., 2006 Biotechol. Bioeng. 353—61), level of acidity of the culture (see e.g., Kobayashi H., et al., 1997 FEMS Microbiol Lett 152(2):235—42), concentrations of substrates and/or ions (see e.g., Ko JH. et al., 2996 Appl Biochem hnol 60(1):41—8). In addition, yeast systems can be used to produce antibodies with an extended half-life (see e.g., Smith, BJ. et a1. 2001 Bioconjug Chem 12(5):750-756), In one preferred embodiment, antibodies are produced in mammalian cells.

Preferred mammalian host cells for expressing the clone antibodies or antigen—binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr— CHO cells, described in Urlaub and , 1980, Proc. Natl. Acad. Sci. USA 77:4216— 4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Irnmunol. Methods (2004) 289(1—2):65—80), and a cell from a transgenic animal, e.g., a transgenic mammal. For e, the cell is a mammary epithelial cell.

In some embodiments, plasma kallikrein binding antibodies are produced in a plant or cell—free based system (see e.g., Galeffi, P., et al., 2006 J Transl Med 4:39).

In addition to the nucleic acid ce encoding the diversified immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates ion of host cells into which the vector has been introduced (see e.g., US. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or rexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate ase (DHFR) gene (for use in dhfr' host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

In an ary system for recombinant expression of an antibody, or antigen— binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr' CHO cells by calcium phosphate—mediated transfection. Within the inant expression vector, the antibody heavy and light chain genes are each ively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of ription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate ion/ampli?cation. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology ques are used to prepare the recombinant sion vector, ect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

For antibodies that include an Fc domain, the antibody production system may produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary—type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by ch receptors and complement Clq (Burton and Woof, l992, Adv. Immunol. 51:1— 84; Jefferis et al., 1998, Immunol. Rev. 163259—76). In one embodiment, the Fc domain is produced in a mammalian sion system that appropriately glycosylates the residue corresponding to gine 297. The PC domain can also include other eukaryotic post-translational modi?cations.

Antibodies can also be produced by a transgenic animal. For e, US.

Pat. No. 5,849,992 describes a method of expressing an antibody in the y gland of a transgenic mammal. A transgene is constructed that includes a milk- specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for ion. The milk ed by females of such transgenic mammals includes, secreted—therein, the antibody of interest. The antibody can be purified from the milk, or for some ations, used directly.

An dy as bed herein (e.g., DX-2930) can be present in a composition, 2. g. a pharmaceutically acceptable composition or pharmaceutical ition. The antibody as described herein (e.g., DX—2930) can be formulated together with a pharmaceutically able carrier. In some embodiments, 30 mg— 400 mg of DX—2930 antibody are present in a composition, ally with a pharmaceutically acceptable carrier, (e.g., a pharmaceutically acceptable composition or pharmaceutical composition. In some embodiments, 30 mg, 100 mg, 150 mg, 300 mg, or 400 mg of DX-2930 antibody are present in a composition optionally with a pharmaceutically acceptable carrier, e.g., a pharmaceutically able composition or pharmaceutical composition.

A pharmaceutically acceptable carrier es any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption ng agents, and the like that are logically compatible. Preferably, the carrier is suitable for subcutaneous, intravenous, intramuscular, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), although rs suitable for inhalation and intranasal stration are also contemplated. In some ments, the pharmaceutically acceptable carrier is one or more of sodium phosphate, citric acid, histidine, sodium chloride, and Tween 80. In some embodiments, the pharmaceutically able carrier is sodium phosphate, citric acid, histidine, sodium chloride, and Tween 80. In some embodiments, the antibody, such as DX—2930, is formulated in 30 mM sodium phosphate, 8.6 mM citric acid, 50 mM histidine, 90 mM sodium chloride, 0.01% Tween 80, pH 6.0. In some embodiments, the composition comprises or consists of 100 mg DX—2930 per 1 mL solution of 30 mM sodium phosphate, 8.6 mM citric acid, 50 mM histidine, 90 mM sodium chloride, 0.01% Tween 80.

A pharmaceutically acceptable salt is a salt that retains the desired biological activity of the compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977, J. Pharm. Sci. 66: 1—19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic nic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono— and dicarboxylic acids, phenyl—substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts e those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as N,N'—dibenzylethylenediamine, N—methylglucamine, chloroprocaine, choline, diethanolamine, nediamine, ne, and the like.

The compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, mes and suppositories. The form can depend on the ed mode of administration and therapeutic application. Many compositions are in the form of injectable or ble solutions, such as compositions similar to those used for administration of humans with antibodies. An exemplary mode of stration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the plasma kallikrein binding protein is administered by intravenous infusion or injection.

In another preferred embodiment, the plasma kallikrein binding protein is administered by intramuscular or subcutaneous injection. In another preferred embodiment, the plasma kallikrein binding protein is administered by eritoneal injection.

The phrases “parenteral stration” and “administered parenterally” as used herein means modes of administration other than enteral and l administration, usually by ion, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraar'ticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the binding protein in the ed amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, ed by ?ltered sterilization. lly, dispersions are prepared by incorporating the active compound into a e vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of ation are vacuum drying and freeze—drying that yields a powder of the active ingredient plus any onal desired ingredient from a previously sterile—filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of tants. Prolonged absorption of injectable itions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

An antibody as described herein (e.g., DX-2930) can be administered by a variety of methods, including intravenous injection or infusion. For e, for some therapeutic applications, the antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or 7 to 25 mg/m2. The route and/or mode of administration will vary depending upon the desired results. In certain ments, the active compound may be prepared with a carrier that will protect the nd against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and ctic acid. Many methods for the ation of such formulations are available. See, e. g., Sustained and Controlled Release Drug Delivery Systems, J .R.

Robinson, ed., 1978, Marcel Dekker, Inc., New York. ceutical compositions can be administered with medical devices. For example, in one embodiment, a pharmaceutical composition disclosed herein can be administered with a device, e.g., a needleless hypodermic injection device, a pump, or implant.

In certain embodiments, an antibody as described herein (e.g., DX—2930) can be formulated to ensure proper distribution in vivo. For e, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the eutic compounds disclosed herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., US. Pat. Nos. 4,522,811; 548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus e targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin.

Pharmacol. 29:685).

Dosage ns are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or sed as indicated by the cies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the d therapeutic effect in ation with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular eutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An ary, non—limiting range for a therapeutically or prophylactically effective amount of an antibody as described herein (e.g., DX—2930) is 30 mg to 400 mg, or any integer in between, for example, 100—400 mg, 100—300 mg, or 300—400 mg. In some embodiments, the therapeutically or prophylactically effective amount is mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg.

In some embodiments, the therapeutically or prophylactically effective amount of an antibody described herein (e.g., DX—2930) is 30 mg, 100 mg, 150 mg, 300 mg, or 400 mg. In some ments, the therapeutically or prophylactically ive amount is 150 mg. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg. In some embodiments, the therapeutically or prophylactically effective amount is 400 mg.

In some embodiments, the therapeutically or prophylactically effective amount is administered at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more. In some embodiments, the therapeutically or prophylactically effective amount is administered every other week (i.e., every two weeks). In some embodiments, the therapeutically or prophylactically effective amount is 300 mg or 400 mg and the amount is administered every two weeks. In some embodiments, the eutically or prophylactically effective amount is 300 mg and this amount of the antibody is stered every two weeks. In some embodiments, the therapeutically or prophylactically effective amount is 400 mg and this amount of the antibody is administered every two weeks.

In some embodiments, treatment of any of the anti—pKal antibody such as DX— 2930 involves a treatment regimen comprising at least a loading period and a maintenance period. In some embodiments, the therapeutically or prophylactically effective amount of the antibody for the loading period is 100 to 300 mg (e.g., 150 mg or 300 mg) per each stration. During this period, the antibody may be administered every week (e.g., every week for one, two or three weeks). In one example, the loading period is 2 weeks and the antibody is administered at day 0, day 7, and day 14.

Alternatively or in addition, the therapeutically or prophylactically effective amount for the nance period is about 100 to 300 mg (e.g., 150 mg or 300 mg) per each administration. During this period, the antibody can be administered every other week (i. 6., every two weeks), every three weeks, or every four weeks (e.g., every two weeks for ten weeks, resulting in delivery of 5 doses total). In one example, the maintenance period may last for 10 weeks and the antibody is administered at day 28, day 42, day 56, day 70, and day 84.

In some embodiments, the anti—pKal dy such as DX—2930 is administered at 150 mg or 300 mg and the amount is first administered every week for a suitable period (e.g., every week for one, two or three weeks) and subsequently stered every two to four weeks (e.g., every two, three or four weeks) for a suitable period.

In any of the methods described herein, the treatment regimen may further comprise a follow-up period after the maintenance period. In the follow—up period, the antibody such as DX-2930 may be administered every 2-4 weeks at 100-300 mg, for example 300 mg. In some instances, the dosage may increase to 400 mg in one or more of the g period, the maintenance period, and the follow—up .

The pharmaceutical compositions disclosed herein may include a “therapeutically ive amount” or a “prophylactically effective amount” of an antibody as described herein (e.g., DX—2930).

An antibody as described herein (e.g., DX-2930) can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) a DX—2930 antibody, e.g., a composition (e.g., a pharmaceutical composition) that includes the antibody, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to a method described herein and/or the use of an antibody as described herein (e.g., DX—2930), e.g., for a method described herein. In some embodiments, the kit comprises one or more doses of DX- 2930. In some embodiments, the one or more doses are 30 mg, 100 mg, 150 mg, 300 mg or 400 mg.

The informational al of the kit is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to using the antibody to treat, prevent, or diagnosis of disorders and conditions, e.g., a plasma kallikrein associated disease or condition.

In one embodiment, the informational al can include instructions to administer n antibody as described herein (e.g., DX—2930) in a suitable manner to perform the methods bed herein, e.g., in a suitable dose, dosage form, mode of administration or dosing schedule (e.g., a dose, dosage form, dosing schedule or mode of administration described herein). In another ment, the informational material can include ctions to administer an antibody as described herein (e.g., DX—2930) to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a plasma kallikrein associated e or condition. For example, the al can include instructions to administer an antibody as bed herein (e.g., DX—2930) to a t with a disorder or condition described herein, e.g., a plasma kallikrein associated e, e.g., ing to a dosing schedule described . The informational material of the kits is not limited in its form. In many cases, the ational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.

An antibody as described herein (e.g., 0) can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that an antibody be substantially pure and/or sterile. When an antibody is provided in a liquid solution, the liquid on preferably is an aqueous solution, with a sterile aqueous solution being preferred. When an antibody is provided as a dried form, reconstitution generally is by the addition of a suitable t. The solvent, e.g., sterile water or buffer, can optionally be ed in the kit.

The kit can include one or more containers for the composition containing an antibody as bed herein (e.g., DX—2930). In some ments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, Vial, or syringe, and the informational material can be ned in association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the ition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an antibody as described herein (e.g., DX—2930). For example, the kit includes a ity of es, ampules, foil packets, or blister packs, each containing a single unit dose of an antibody as described herein (e.g., DX—2930). The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light—tight.

The kit optionally includes a device suitable for administration of the composition, e.g., a e, or any such delivery device. In one embodiment, the device is an implantable device that dispenses metered doses of the antibody. The disclosure also features a method of providing a kit, e.g., by combining components described herein.

Treatment In some aspects, the disclosure provides the use of an dy as described herein (e.g., DX-2930) in ng HAE.

Hereditary Angioedema Hereditary angioedema (HAE) is also known as “Quincke edema,” Cl esterase inhibitor deficiency, Cl inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent es of severe swelling (angioedema), which can affect, e.g., the limbs, face, genitals, gastrointestinal tract, and airway. Symptoms of HAE include, e.g., swelling in the arms, legs, lips, eyes, tongue, and/or throat; airway ge that can involve throat swelling and sudden ness; repeat episodes of abdominal cramping without obvious cause; and/or swelling of the intestines, which can be severe and can lead to abdominal cramping, vomiting, dehydration, diarrhea, pain, and/or shock. About one—third of individuals with this HAE develop a non-itchy rash called erythema marginatum during an attack.

Swelling of the airway can be life threatening and causes death in some ts. Mortality rates are estimated at 15-33%. HAE leads to about 15,000-30,000 emergency ment visits per year.

Trauma or , e.g., dental procedures, sickness (e.g., viral illnesses such as colds and the ?u), menstruation, and surgery can r an attack of angioedema. To prevent acute s of HAE, patients can attempt to avoid specific stimuli that have previously caused attacks. r, in many cases, an attack occurs without a known trigger. Typically, HAE ms first appear in childhood and worsen during puberty. On average, untreated individuals have an attack every 1 to 2 weeks, and most episodes last for about 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 111, all of which can be treated by the methods described herein. It is estimated that HAE s l in 50,000 people, that type I accounts for about 85 percent of cases, type II accounts for about percent of cases, and type III is very rare. Patients having type I or type II HAE are typically deficient in Cl—INH. Such patients either have a defective Cl—INH gene and thus do not produce Cl-INH, or produce al Cl—INH proteins. Type III is the most newly described form and was originally thought to occur only in women, but families with affected males have been identified. Type III HAE is believed to be unassociated with Cl-INH. Patients having type III HAE may have normal Cl-INH proteins.

HAE is inherited in an autosomal dominant pattern, such that an affected person can inherit the mutation from one affected parent. New ons in the gene can also occur, and thus HAE can also occur in people with no history of the disorder in their family. It is estimated that 20—25% of cases result from a new spontaneous mutation.

Mutations in the SERPINGl gene cause hereditary angioedema type I and type II. The SERPINGl gene provides instructions for making the C1 inhibitor protein, which is ant for controlling ation. C1 inhibitor blocks the activity of certain proteins that promote in?ammation. Mutations that cause hereditary angioedema type I lead to reduced levels of Cl inhibitor in the blood. In contrast, mutations that cause type II result in the production of a C1 inhibitor that functions abnormally. t the proper levels of functional C1 inhibitor, excessive s of bradykinin are generated. Bradykinin promotes in?ammation by increasing the leakage of ?uid through the walls of blood vessels into body tissues. ive accumulation of ?uids in body tissues causes the episodes of swelling seen in individuals with hereditary dema type I and type II.

Mutations in the F12 gene are associated with some cases of hereditary angioedema type III. The F12 gene provides instructions for making coagulation factor XII. In addition to g a critical role in blood clotting lation), factor XII is also an important stimulator of in?ammation and is involved in the production of bradykinin. Certain mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is generated and blood vessel walls become more leaky, which leads to episodes of ng. The cause of other cases of hereditary angioedema type III remains unknown. Mutations in one or more as—yet unidentified genes may be sible for the disorder in these cases.

HAE can present similarly to other forms of angioedema resulting from allergies or other medical conditions, but it differs significantly in cause and treatment. When hereditary angioedema is misdiagnosed as an allergy, it is most ly treated with antihistamines, steroids, and/or hrine, which are typically ineffective in HAE, although epinephrine can be used for life—threatening reactions. Misdiagnoses have also resulted in ssary exploratory surgery for patients with abdominal swelling, and in some HAE patients abdominal pain has been incorrectly diagnosed as psychosomatic.

C1 inhibitor therapies, as well as other therapies for HAE, are described in , A.P., J Allergy Clin Immunol, 2010, 126(5):918—925.

Acute treatment of HAE attacks is provided to halt progression of the edema as quickly as possible. C1 inhibitor concentrate from donor blood, which is administered intravenously, is one acute treatment; however, this treatment is not available in many countries. In emergency situations where Cl inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as an alternative, as it also contains Cl inhibitor.

Purified Cl inhibitor, d from human blood, has been used in Europe since 1979. Several Cl inhibitor treatments are now available in the U.S. and two Cl inhibitor products are now available in Canada. Berinert P (CSL Behring), which is pasteurized, was approved by the FDA. in 2009 for acute attacks. Cinryze (ViroPharma), which is ltered, was ed by the FDA. in 2008 for prophylaxis. Rhucin (Pharming) is a recombinant Cl inhibitor under development that does not carry the risk of infectious disease transmission due to human blood—bome pathogens.

Treatment of an acute HAE attack also can include medications for pain relief and/or IV ?uids.

Other treatment ties can stimulate the synthesis of Cl inhibitor, or reduce Cl inhibitor ption. Androgen medications, such as danazol, can reduce the frequency and severity of attacks by stimulating production of Cl inhibitor.

Helicobacter pylori can trigger abdominal attacks. otics to treat h. pylori will decrease abdominal attacks.

Newer ents attack the contact cascade. Ecallantide (KALBITOR®, DX— 88, Dyax) ts plasma kallikrein and has been approved in the U.S.. Icatibant (FIRAZYR®, Shire) inhibits the bradykinin B2 receptor, and has been approved in Europe and the U.S.

Diagnosis of HAE can rely on, e.g., family history and/or blood tests.

Laboratory findings associated with HAE types I, II, and III are described, e. g., in Kaplan, A.P., J Allergy Clin Immunol, 2010, l26(5):9l8—925. In type IHAE, the level of Cl inhibitor is decreased, as is the level of C4, whereas Clq level is normal.

In type II HAE, the level of Cl inhibitor is normal or increased; however, Cl inhibitor on is abnormal. C4 level is decreased and Clq level is . In type III, the levels of Cl inhibitor, C4, and Clq can all be normal.

Symptoms of HAE can be assessed, for example, using questionnaires, e.g., onnaires that are completed by patients, clinicians, or family members. Such questionnaires are known in the art and include, for example, visual analog scales.

See, e.g., McMillan, C.V. et al. Patient. 2012;5(2):ll3—26.

Treating HAE with Kal antibodies The disclosure provides methods of ng (e.g., ameliorating, stabilizing, or eliminating one or more ms) of hereditary angioedema (HAE) by stering an antibody described herein (e.g., a therapeutically effective amount of an antibody described herein) to a subject having or suspected of having HAE, e.g., according to a dosing schedule bed herein. Additionally provided are methods of treating HAE by administering an antibody described herein (e. g., a therapeutically effective amount of an antibody described ), e.g., according to a dosing schedule described , or in combination with a second therapy, e.g., with one other agent, e.g., described herein. The disclosure also provides methods of preventing HAE or a symptom thereof by administering an antibody described herein (e.g., a prophylactically effective amount of an antibody described ) to a subject at risk of developing HAE (e. g., a subject having a family member with HAE or a genetic predisposition thereto), e. g., ing to a dosing schedule described .

In some examples, the subject may be a human patient who has no HAE symptoms at the time of the treatment.

Treating includes stering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a disease or condition.

Methods of administering DX—2930 antibodies are also described in “Pharmaceutical Compositions.” Suitable dosages of the antibody used can depend on the age and weight of the t and the particular drug used. The antibody can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between plasma kallikrein and its substrate (e.g., Factor XII or HMWK). The dose of the antibody can be the amount sufficient to block 90%, 95%, 99%, or 99.9% of the activity of plasma kallikrein in the patient, especially at the site of disease. This may require 30 mg, 100 mg, 300 mg, or 400 mg, e.g., administered every two weeks.

In one embodiment, the antibodies are used to inhibit an activity (e.g., inhibit at least one activity of plasma kallikrein, e.g., reduce Factor XIIa and/or bradykinin production) of plasma kallikrein, e.g., in vivo. The binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope.

The antibodies can be used directly in vivo to eliminate antigen—expressing cells via natural complement—dependent cytotoxicity (CDC) or antibody dependent cellular xicity (ADCC). The antibodies described herein can include complement binding effector , such as the Fc portions from IgGl, —2, or —3 or corresponding portions of IgM which bind complement. In one embodiment, a population of target cells is ex vivo treated with an antibody described herein and appropriate effector cells. The treatment can be supplemented by the addition of ment or serum containing complement. Further, phagocytosis of target cells coated with an antibody described herein can be improved by binding of complement proteins. In another ment target, cells coated with the antibody which includes a complement binding effector domain are lysed by complement.

Methods of administering DX—2930 antibodies are described in “Pharmaceutical Compositions.” Suitable s of the molecules used will depend on the age and weight of the subject and the ular drug used. The dies can be used as competitive agents to inhibit or reduce an undesirable interaction, e.g., between a natural or pathological agent and the plasma kallikrein.

A therapeutically effective amount of an antibody as described , can be stered to a subject having, suspected of having, or at risk for HAE, thereby treating (e.g., ameliorating or improving a symptom or feature of a disorder, slowing, stabilizing and/or halting disease ssion) the disorder.

The antibody described herein can be administered in a therapeutically effective amount. A eutically effective amount of an antibody is the amount which is effective, upon single or multiple dose stration to a subject, in treating a subject, e. g., curing, alleviating, relieving or improving at least one symptom of a disorder in a t to a degree beyond that expected in the absence of such treatment.

Dosage regimens can be adjusted to provide the optimum desired se (e. g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to ate parenteral compositions in dosage unit form for ease of stration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to e the d therapeutic effect in association with the required pharmaceutical carrier.

In some embodiments, the DX—2930 antibody is administered by multiple doses such as once every 2 weeks, once every 3 weeks, once every four weeks, once every 6 weeks, once every 8 weeks or less frequent. Each of the multiple doses can be 30 mg, 100 mg, 150 mg, 300 mg, 350 mg. or 400 mg. In some instances, a patient may be given multiple doses once every 2 weeks, for a suitable period of time. In some embodiments, DX—2930 can be administered at 300 mg or 400 mg every two weeks. In other embodiments, DX—2930 can be administered at 300 mg or 400 mg every four weeks. In yet other embodiments, DX—2930 can be administered at 150 mg every four weeks. In any of the methods described herein, a subject treated by DX—2930 for le doses as described herein may be followed up with a maintenance treatment.

In any of the methods described herein, a t may be d by DX—2930 for multiple doses in a loading period and then followed up with a maintenance period. In the loading period, the subject may be treated with DX—2930 at about 100 mg to about 400 mg (e.g., 100—300 mg or 150-300 mg, for example, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg) once every 2—4 weeks (for example, every 2 weeks, every 3 weeks, or every 4 weeks) for a suitable period. In the loading period, the t may be treated with DX-2930 at about 100 mg to about 300 mg (e.g., 100—300 mg or 150—300 mg, for example, 100 mg, 150 mg, 200 mg, or 300 mg) once every 2—4 weeks (for example, every 2 weeks, every 3 weeks, or every 4 weeks) for a le period.

In some embodiments, the patient can be monitored for side effects (e.g., elevation of creatine atase levels) and/or inhibition levels of pKal by the antibody (e.g., serum or plasma concentration of the antibody or the pKal activity level) before and after the ent or during the course of treatment. If adverse effect is observed, the dose of the antibody might be reduced or the treatment might be terminated. If the inhibition level is below a minimum therapeutic level, further doses of the antibody might be administered to the patient.

In some embodiments, the plasma or serum concentration of the dy (e.g., DX—2930) may be measured during the course of the treatment (e.g., after the l dosage) for assessing the efficacy of the treatment. If the plasma or serum concentration of the antibody is lower than about 80 nM, a follow—up dosage may be needed, which may be the same or higher than the initial dosage. The plasma or serum concentration of the antibody may be measured by determining the protein level of the antibody in a plasma or serum sample ed from the subject, e.g., by an immune assay or MS assay. The plasma or serum concentration of the antibody may also be measured by determining the tory level of pKal in a plasma or serum sample obtained from a subject treated with the antibody. Such assays may include the synthetic substrate assay or the Western blot assay for measuring cleaved kininogen as described herein.

Alternatively or in addition, the plasma or serum level of creatine kinase can be monitored during the course of the treatment. If the plasma or serum level of creatine kinase is found to elevate during the ent, the dosage of the antibody may be reduced or the treatment may be terminated.

In some embodiments, an optimal dosage (e.g., optimal prophylactic dosage or optimal eutic dosage) of the antibody (e.g., DX—2930) may be determined as s. The antibody is given to a t in need of the treatment at an initial dose.

The plasma concentration of the antibody in the subject is measured. If the plasma concentration is lower than 80 nM, the dose of the antibody is increased in a subsequent administration. A dosage of the antibody that maintains the antibody plasma concentration above about 80 nM can be chosen as the optimal dosage for the subject. The creatine phosphokinase level of the subject can be monitored during the course of treatment and the optimal dosage for that subject can be further adjusted based on the creatine phosphokinase level, e.g., the dosage of the dy might be reduced is elevation of creatine phosphokinase is observed during treatment. In some embodiments, the dy such as DX-2930 is administered to reduce the level of cleaved kininogen to levels comparable to healthy subjects.

In some embodiments, any of the antibodies disclosed herein, such as DX— 2930 and its onal variants, may be used to prevent HAE attack or reduce the rate of HAE attack in human patients having history of HAE attack. In some examples, the human patients experienced at least two HAE attacks per year and optionally at least one within the 6 months prior to the ent. In other es, the human patients experienced at least two HAE attacks within 3 months prior to the treatment.

In other examples, the human patients had at least 9 HAE attacks within 3 months prior to the treatment and optionally at least 25 attacks (e.g., 36 attacks) within 12 months prior to the treatment.

An antibody as bed herein (e.g., DX-2930) can be administered in combination with one or more of the other therapies for treating a disease or condition associated with plasma kallikrein activity, e.g., a disease or condition described herein. For example, an antibody as described herein (e.g., DX—2930) can be used therapeutically or prophylactically with surgery, another anti- plasma kallikrein Fab or IgG (6.g. another Fab or IgG described herein), another plasma kallikrein inhibitor, a peptide inhibitor, or small molecule inhibitor. es of plasma kallikrein inhibitors that can be used in combination y with a plasma kallikrein binding antibodies bed herein include plasma kallikrein inhibitors described in, e. g. WO 95/21601 or One or more plasma kallikrein inhibitors can be used in combination with an antibody as described herein (e.g., DX—2930). For example, the combination can result in a lower dose of the inhibitor being needed, such that side effects are reduced.

An antibody as described herein (e.g., 0) can be administered in combination with one or more current therapies for treating HAE. For example, DX— 2930 antibody can be d with a second anti—HAE therapeutic agent such as ecallantide, a Cl esterase inhibitor (e.g., CINRYZETM), aprotinin (TRASYLOL®), and/or a bradykinin B2 receptor inhibitor (6.3., icatibant (FIRAZYR®)).

The term “combination” refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or ies overlaps in time. The agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two te formulations administered concurrently) or sequentially in any order. Sequential administrations are strations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks. The use of a plasma kallikrein binding antibody described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side effects associated with another agent that is being stered. Accordingly, a ation can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the plasma kallikrein binding antibody.

A combination therapy can include administering an agent that reduces the side s of other therapies. The agent can be an agent that reduces the side effects of a plasma kallikrein associated disease treatment.

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

EXAMPLES Example 1: A Phase 1b, Double-Blind, Multiple Ascending Dose Study to Assess Safety, Tolerability and Pharmacokinetics of 0 in Hereditary Angioedema Subjects A phase lb trial was conducted to assess safety and tolerability of le subcutaneous administrations of DX—2930 at ent dose levels in hereditary angioedema (HAE) subjects. HAE patients included in the study included those with documented diagnosis of HAE (Type I or Type 11) based upon all of the following: documented clinical history consistent with HAE (subcutaneous or mucosal, ritic swelling episodes without accompanying ria), Cl inhibitor (Cl— INH) antigen or functional level < 40% of the normal level (Subjects with antigen or functional Cl-INH level 40-50% of the normal level were enrolled if they also had a C4 level below the normal range and a family history consistent with HAE Type I or II), age at reported onset of first angioedema ms 3 30 years or a family history consistent with HAE Type I or II, and experiencing 22 HAE s per year, with at least 1 attack in the past 6 months reported by the subject. HAE patients ed were randomized in a 2:1 ratio of active drug to placebo and administered subcutaneous doses of DX—2930 at 30 mg (n24), 100 mg (n24), 300 mg (n=5), and 400 mg (n=l l) or placebo (n=l3) in two doses separated by 14 days. One patient in the 400 mg group ed only one dose and then was unavailable for the second dose and was ed. The replacement patient was also unable to complete the study for reasons not related to the study, resulting a total of 10 patients completing the 400 mg dose and being included in the assessment. Plasma was collected at time points following stration out to day 120 (15 weeks), except in the group that received the 400 mg dose, where data was available up to day 50. Analyses ed safety, pharmacokinetics, pharmacodynamics (biomarkers), and efficacy. The drug group and the placebo group were baled in terms of age, race, ethnicity, and BMI, although there were ly more females present in the DX-2930 (67%) vs placebo (54%) groups.

Pharmacokinetics of DX—2930 DX—293O drug levels were measured in plasma by a validated immunoassay that used an anti—idiotypic antibody against DX—2930 D02). From the plot of mean plasma drug levels for each dose group versus days following DX—293O administration, it was evident that drug levels were dose dependent and exhibited a prolonged half—life, typical of a human monoclonal antibody. Key pharmacokinetic observations are summarized in Table 2 and Figure 1. First, Cmax drug levels increased with increasing dose, as expected. In addition, the Tmax following the second dose was about 20 days and the half—life was approximately 14 days. These parameters were consistent with values obtained in a phase la study in healthy eers and supported either once or twice a month dosing.

Table 2. Pharmacokinetic observations 17.9(1.1) 14.2 (0.8) 18.0 (0.5) 14.6 (3.4) 18.2(1.5) 13.8 (3.3) .0) 15.0 (2.4) Values presented are the mean values for the evaluated patients with the standard deviation in parentheses.

Pharmacodynamic Activity of DX—2930: Fluorogenic ty Assay Two different biomarker assays were used to investigate the pharmacodynamic (PD) ty of DX—2930 in HAE patient plasma. The first PD assay is referred to as the Fluorogenic Activity assay, and it provides a measure of the bioactivity of DX—2930 in citrated plasma obtained from treated patients at the same time points following dosing as used to determine the pharmacokinetic properties of DX-2930. This assay measured the amount of active plasma rein that is generated in d plasma after activation of the contact pathway. Specifically, dilute plasma was spiked with active Factor XIIa (FXIIa) which propagates the enzymatic cascade of the contact pathway, after 2 minutes the FXIIa inhibitor Corn Trypsin tor was added to stop the reaction, and the amount of active plasma kallikrein present in the sample was ed by its y to hydrolyze a pro— fluorescent synthetic e substrate. The presence of increasing levels of DX—293O in the plasma of treated healthy volunteers (phase 1a study) or HAE patients (phase lb study) was associated with a dose dependent reduction in the observed plasma kallikrein enzymatic rate (Figure 2). The percent inhibition observed at each time point was calculated relative to the amount of plasma kallikrein activity in the pre— dose samples for each patient. The observed bioactivity in Figure 2 was well correlated with the pharmacokinetic properties of DX—2930 in Figure l. Marked inhibition was ed in the 300 and 400 mg doses following dosing. Intermediate inhibition was ed in the 100 mg dose and no apparent inhibition was evident in the 30 mg dose.

Pharmacodynamic Activity of DX—2930: Western Blot Assay HAE patients are deficient in Cl—Inhibitor, the endogenous inhibitor of plasma kallikrein. As a result, these patients have elevated active plasma kallikrein, which converts its 1-chain high molecular weight kininogen ate to 2-chain and bradykinin, the key or of pain and edema in HAE. DX-2930 is a highly potent inhibitor of active plasma kallikrein that blocks 2-chain and bradykinin generation.

A Western blot assay was developed to measure the relative amounts of 1— chain and 2—chain high molecular weight kininogen in plasma in different plasma anti— coagulants and treatment conditions. Figure 3 shows the %2—chain that was ed in HAE patient plasma collected in the presence of protease inhibitors (SCAT169 plasma). The presence of 9 plasma prevented the activation of the contact system and subsequent 2—chain generation that can occur during blood collection and sing to plasma. Hence, this level of 2—chain was expected to closely match that of endogenous levels in HAE patients in in the phase lb trial (27%) and in healthy volunteers (12%). DX—2930 treated HAE patients exhibited lower 2—chain levels as measured in samples collected either 8 or 22 days after .

In Figure 4, pre-dose citrated plasma obtained from the HAE ts in the phase 1b trial contained approximately 52% 2-chain. In contrast, citrated plasma samples from healthy volunteers obtained in a phase 1a study contained approximately 8% 2—chain. Mean 2—chain levels in plasma of phase lb subjects collected on days 8 and 22 were also investigated and are shown in Figure 4. The statistically significant ion in 2—chain levels in the 300 and 400 mg dose groups versus pre—dose levels demonstrated pharmacodynamic activity of DX—2930.

Figure 5 shows that citrated plasma from DX—2930 dosed HAE patients exhibited less 2—chain following ex vivo tion with coagulation factor XIIa (FXIIa). The 300 and 400 mg dose groups reduced the amount of 2—chain to a level below that observed in healthy volunteers. This ex vivo activation may be considered an in vitro model of a severe HAE .

Both of these PD biomarker assays supported dose selection that would achieve drug levels obtained at the 300 and 400 mg dose groups.

Safety A summary of adverse events is shown in Table 3 below. There was no imbalance in treatment emergent adverse events (TEAEs) that would indicated a safety concern of DX—2930. Most common AEs were HAE attacks, injection site pain, and headache. There were 3 severe TEAEs tion site pain lasting 1 min, worsening headache lasting 1 minute and night sweats). No safety s were identified for clinical laboratory abnormalities or s from baseline, vital signs or physical examinations, or abnormalities or changes in electrocardiogram (ECG).

These results suggest that DX—2930 appears to be well tolerated in HAE patients at doses up to 400 mg.

Table 3. Summary of treatment adverse events - DX-2930 DX-2930 DX-2930 14 (58%) 10 (77%) Deaths or subject discontinuations 2 5 (21%) 5 (39%) Related 1 2 4 7 (29%) 5 (39%) TEAEs** * TEAE: Treatment Emergent Adverse . An AB is treatment emergent if the onset time is after administration of study drug through the Day 120 post dose final follow—up Visit, or in the event that onset time precedes study drug administration, the AE increases in severity during the 120 day post dose follow—up period. ** Treatment—related AEs: Relatedness of ABS to study drug was assessed by a blinded investigator.

Immunogenicity patients were anti—drug antibody positive (2 of the 5 had intermittent, ?uctuating results). r, no positive s were neutralizing, there was no clinical evidence of hypersensitivity, and no apparent effects on pharmacokinetics or biomarkers.

Efficacy assessment A prospective primary efficacy analysis was performed, which focused on the 300 and 400 mg doses of DX-2930, individually and combined, compared to placebo.

A six week primary ment period (Day 8 to Day 50) was used (Figure 6A and 6B), as PK modeling from phase 1a ted e drug exposure during this time interval. The primary analysis focused upon subjects with a minimum baseline history of at least 2 attacks in the past 3 months. Most of the subjects fulfilled the minimum ed baseline y of at least 2 attacks in the past 3 months. Of the 13 placebo subjects, 11 met this requirement. Of the 16 subjects treated with 300 or 400 mg DX—2930, 15 met this requirement. The ne HAE attack rates in the o, 300 mg, and 400 mg groups were 0.39, 0.33, and 0.55 attacks per week, respectively.

The baseline rate in the combined 300 and 400 mg group was 0.49 attacks per week.

The primary approach was an intent—to—treat (ITT) analysis. A model of repeated measurements was used, with an analysis of variance (ANOVA) employing baseline attack rates as a ate. The read—out was expressed as a percent reduction in HAE attacks by DX-2930 in comparison to the placebo attack rates, and p values were calculated.

Results of the efficacy assessment are shown in Table 4 and 5 and in Figure 7, which both show a reduction in HAE attack rate with the 300 and 400 mg doses of DX—2930, individually and combined, compared to the placebo. In particular, 13 of the 15 DX—2930 subjects treated with 300 or 400 mg were attack—free for the duration of the study, whereas for the placebo group, only 3 of the 11 subjects were attack—free.

Table 4. Reduction in HAE attack rate from day 8 to 50 DX-2930 DX-2930 DX-2930 300 mg 400 mg Combined (N: 4) (N: 11) 300 and 400 mg P—value vs placebo <0.0001 0.005 0.0012 Note: Only subjects who have a baseline attack rate of at least 2 s in the last 3 months were ed.

* Mixed Model Repeated Measurements with Analysis of Variance (baseline attack frequency as ate) and assuming Poisson bution.

Table 5. Proportion of subjects who were attack-free DX-2930 DX-2930 DX-2930 300 mg 400 mg Combined (N = 4) (N = 11) 300 and 400 mg = 15) Attack-free 4/4 (100%) 9/11 (82%) 13/15 (87%) 3/11 (27%) subjects (Day 8 to p = 0.026 p = 0.030 p = 0.004 Among the placebo subjects, there were a total of 24 attacks during the primary efficacy assessment period. Of these 24 attacks, the primary attack location was abdominal in 13 and laryngeal in l of them. 10 of the attacks were severe and 6 were moderate. Acute treatment for the attacks was received in 22 of the 24 attacks.

Among subjects treated with 300 or 400 mg DX—2930, one subject treated with 400 mg had a single HAE attack. This attack was peripheral, mild, lasted 8 hours, and did not require any acute ent. The other 400 mg DXtreated subject experiencing s had two peripheral attacks. One attack was moderate and the other was severe; both attacks were treated with acute therapy. A summary of the characteristics of the HAE s is shown in Table 6.

Table 6. Characteristics of HAE attacks (Day 8 to 50) DX-2930 DX-2930 300 mg 400 mg (N = 4) (N = 11) Acute s requiring 0 2 treatment Next, a modified intent—to—treat (mITT) oc analysis was undertaken.

The ITT population was used except 2 subjects were excluded (one subject that did not receive 2 strations and 1 subject that did not have HAE type 1 or type 2).

Results of the modified analysis are shown in Table 7. In this modified intent—to—treat analysis, from Day 8 to Day 50 in ison to placebo, the 300 mg DX—2930 group had a 100% reduction in s with a p value of less than 0.0001. The 400 mg DX— 2930 group had a 95% reduction in attacks with a p value of 0.0022. The combined 300 and 400 mg DX—2930 group had a 97% reduction in HAE attacks, with a p value Table 7. y efficacy analysis (Day 8 to 50) (modified intent-to-treat analysis) DX-2930 DX-2930 DX-2930 300 mg 400 mg Combined (N: 4) N=9 300 and 400 mg (N: 13) P—value vs placebo <0.0001 0.0022 0.0007 Note: Only subjects who have a baseline attack rate of at least 2 attacks in the last 3 months are ed. MITT population excludes 2 subjects, one t t type I or II HAE and one subject who received only 1 administration of DX—2930.

* Mixed Model Repeated ements with Analysis of Variance (baseline attack frequency as covariate) and ng Poisson distribution.

The incidence of HAE attacks in relation to drug exposure over time was also evaluated. Without wishing to be bound by theory, the hypothesis was that higher drug levels should be correlated with prevention of HAE attacks, meaning (a) that before dosing as well as in the few days after dosing, attacks should be observed, (b) that as drug levels accumulate, attacks should become rare or even absent, and (c) that as the drug levels decline, attacks should re—emerge. The results of this evaluation are shown in Figures 8-13.

In the placebo group high incidence of attacks was evident (Figure 8). These events were distributed throughout the entire duration of the study without any particular pattern.

In the 30 and 100 mg 0 groups, no attacks occurred from Day 8 to Day 50 (Figures 9 and 10). However, the baseline attack rates for these groups were relatively lower and the pharmacodynamic effect for the 30 mg group was not appreciably different from that for placebo. In the 300 mg DX—2930 group, there were attacks that occurred prior to . As drug levels rose, the subjects became attack—free and as drug levels declined, attacks re-emerged (Figure 11). A r pattern was also observed in the 400 mg DX—2930 group (Figure 12). Attack incidence substantially decreased during a period of notable drug exposure, particularly within the Day 8 to Day 50 time interval. This period was bracketed by attacks occurring during times of lower drug exposure, either prior to drug accumulation or as drug levels waned. These results show that there is a clear ation between DX-2930 drug exposure and prevention of HAE attacks.

To further assess the efficacy of 0, the therapeutic effects of DX-2930 were also observed in HAE patients with high baseline attack rates were explored.

Subjects with at least 9 attacks in the past 3 months prior to dosing were identified and evaluated. There were 6 such subjects— 1 treated with placebo, 1 treated with 300 mg DX—2930, and 4 d with 400 mg DX—2930.

In the placebo subject, attacks occurred throughout the observation period at a high rate (Figure 13, panel A). In contrast, the subject treated with 300 mg DX—2930 was attack—free when drug levels were high. (Figure 13, panel B).

In the four subjects with high baseline attack rates who were treated with 400 mg DX—2930, all four of these subjects were attack—free during the Day 8 to Day 50 time interval (Figures 13, panels C—F). This ed one subject with a very high baseline rate of 36 attacks in the past 3 months. The n of attacks in these DX— 2930—treated subjects was consistent with that seen in the 300 and 400 mg groups overall. These individuals did not experience any attacks when drug levels were high.

Attacks only occurred when drug levels were low, either prior to meaningful drug accumulation or after drug levels declined.

From these data, it was observed that the therapeutic effect of 0 was also evident in HAE ts with high baseline attack rates.

In summary, this study shows (a) that there were no apparent safety signals for DX-2930, (b) that the PK profile was consistent overall with a monoclonal dy and ted a regimen of dosing once every 2 weeks or even less frequently, (c) that pharmacodynamic data demonstrated that DX—2930 normalizes the aberrant instability of HAE plasma, at least in the context of the kininogen biomarker , and (d) that a highly statistically significant finding of HAE attack prevention by DX— 2930 was observed. Specifically, in comparison to placebo, there were 100% and 88% reduction in attacks by the 300 and 400 mg DX—2930 treatment groups respectively. This clinical effect was lly associated with drug exposure over time and was also observed in the subset of patients with high baseline attack rates.

These data demonstrate proof of concept for 0 in erm prophylaxis against HAE attacks.

Example 2 : Pharmacodynamic Effect of DX-2930 on Plasma Kallikrein in Hereditary Angioedema Patients Attacks of hereditary angioedema (HAE) result from uncontrolled t system activation which generates a burst of plasma kallikrein (pKal) that cleaves high—molecular—weight kininogen (HMWK) to produce 2—chain HMWK and the inducing peptide, bradykinin. DX—2930 is a human monoclonal antibody inhibitor of pKal in development for the prevention of HAE attacks. The pharmacodynamic bioactivity of DX—2930 was assessed in subjects with HAE.

As described in Example 1 above, the phase lb center, double—blind study, randomized subjects with Type 1 or 2 HAE to receive 2 subcutaneous doses of 0 in dose groups of 30, 100, 300 or 400 mg (n=4, 4, 5, 11) or placebo (n=l3).

Blood samples were obtained prior to and following administration of study drug (Days 1, 8, 22, 64, 92, 120). The ability of DX-2930 to inhibit pKal in basal and FXIIA—activated citrated plasma was assessed using Western blot for 2—chain HMWK.

The s obtained from this study showed that mean 2—chain HMWK levels were significantly reduced and essentially normalized in the 300 and 400 mg dose groups on Days 8 and 22, and on Days 8, 22 and 50, respectively, when compared to placebo d subjects. Treatment with 300 or 400 mg DX—2930 also attenuated the burst in 2—chain generation to levels at or below that observed in healthy individuals in FXIIA—activated samples. Levels of 2—chain HMWK did not differ from pre—dose plasma samples in either ted or inactivated samples collected on Days 64, 92 or 120 ing DX-2930, which correspond to periods of low drug exposure.

In sum, this study indicates that DX-2930 ts pKal in a dose and time- dependent manner in HAE patients.

Example 3 : Relationship between Drug Exposure and Clinical Response Observed in the Phase 1b Study of DX-2930 in Subjects with Hereditary Angioedema DX—2930 is a human monoclonal antibody inhibitor of plasma kallikrein in development for the prevention of tary dema (HAE) attacks. Data from the phase lb study of DX—2930 in HAE subjects as described in Example 1 above was ed to characterize the relationship between drug exposure and al response.

This phase lb multi-center, —blind study, randomized subjects with Type 1 or 2 HAE to receive 2 subcutaneous doses of DX—2930 in dose groups of 30, 100, 300 or 400 mg (n=4, 4, 5, 11) or o (n=13). In this post—hoc analysis, the incidence of HAE attacks was ted in relation to drug exposure over time. In addition, a oc modified intent-to-treat ef?cacy analysis (MITT) was conducted to assess clinical effect in the context of subjects receiving the full dose regimen of DX—2930.

Placebo—treated subjects reported HAE attacks throughout the study (9 subjects, 65 HAE attacks). In the 300 and 400 mg dose groups, HAE attacks were reported prior to or just after initial dosing. When drug levels were high (Day 8 to 50), all but 1 subject was attack—free. As drug levels waned, attacks re—emerged. In the MlTT efficacy analysis, from Day 8 to 50 in comparison to placebo, the 300 and 400 mg DX—2930 groups had a 100% (P < 0.0001) and 95% (P = 0.0022) reduction in attacks, respectively.

Thus, this study indicates that HAE attacks were substantially sed or were eliminated during periods of notable drug exposure consistent with the suggestion that higher drug levels should correlate with HAE attack prevention.

Example 4 : Modeling and Analyses to Identify Potential Dosing Regimens of DX-2930 for the Long-Term Prophylaxis of Hereditary Angioedema DX—2930 is a human monoclonal antibody inhibitor of plasma rein in development for the prevention of hereditary angioedema (HAE) attacks. Data from the Phase 1 studies of DX-2930 as described in Example 1 above were modeled and analyzed to identify potential dosing regimens.

Pharmacokinetic, codynamic and ef?cacy data from the Phase 1 clinical studies were examined. The incidence of HAE attacks was evaluated in relation to plasma drug trations to estimate steady-state trough drug levels necessary to t attacks.

Dosing ns of 300 mg DX—2930 every 2 (q2) or 4 (q4) weeks, and 150 mg q4 weeks are being considered for the l efficacy study. Pharmacokinetic modeling predicts —state trough plasma concentrations of 27,000, 9,500, and 4,750 ng/mL, respectively. In the Phase lb study, at 27,000 ng/mL (corresponding to imate drug levels at Day 22 for 300 mg DX—2930), 2—chain high—molecular— weight gen was suppressed to a level approximating that observed in healthy subjects. Three-hundred mg q2 is therefore predicted to normalize the instability of HAE plasma at steady state. As sful HAE prophylaxis may not require such a high level of pharmacodynamic effect, an analysis of al effect in relation to plasma drug trations was also conducted. In the Phase lb study following DX— 2930 treatment, 24/25 attacks (96%), 21/25 (84%), and 18/25 s (72%) occurred below plasma concentrations of 27,000, 9,500, and 4,750 ng/mL, respectively, suggesting a meaningful range of al response is ated with this range of drug exposure.

In this is, potential dosing regimens of DX—2930 were identified for further clinical investigation in the pivotal efficacy study.

Example 5 : Hereditary Angioedema is Associated with Neuropathic Pain, Systemic Lupus Erythematosis and Systemic Mastocytosis in an Analysis of a Health Analytics Claims Database The plasma kallikrein kinin system (KKS) has been associated with a variety of diseases in addition to being a key mediator of hereditary angioedema (HAE). It was explored in this study whether HAE patients were predisposed to develop such KKS associated diseases: abdominal aortic aneurysm, anaphylaxis, cardiac egia syndrome, Crohn’s disease, diabetic macular edema, idiopathic anaphylaxis, neuropathic pain, psoriasis, psoriatic arthritis, retinopathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), system mastocytosis, systemic vasculitis, thrombotic cerebrovascular accident, and ulcerative colitis.

The Truven MarketScan Database containing individual-level claims data from medical payers and Medicare supplemental plans for 80 million lives in the US. from 1/2010 through 7/2014 was utilized in this study. Within this dataset, an HAE population (n=1063) and 2 control populations: an angioedema population (n=138,851) and the general population (n=79,971,098) exclusive of HAE patients were d using a combination of ICD—9 and prescription drug codes. Claims for comorbid diseases were identified and compared across the populations with calculated odds ratios and 95% confidence intervals (CI).

As shown in the Table 8 below, in the HAE population, SLE was observed 2.3 times more often (OR 2.30, 95%CI: 1.47—3.59) than the angioedema control population. Neuropathic pain was observed 1.45 times (OR 1.45, 95%CI: 1.01—2.09) and ic mastocytosis 4.79 times (OR 4.79, 95%CI: 1.51—15 .18) more often than the angioedema control population.

Table 8. Association between HAE and Other KSS-related Diseases HAE Angioedema Associated Disease Population Control Odds Ratio (95% Abnormal aortic aneurysm 0.56 583 0.42 1.35 (0.60, 3.02) (AAA) Anaphylaxis ‘l15293 11.01 0.72 89) Cardiac egia syndromeN 2.97 (0.73, 12.09) ic r edema --—- 1.87 (0.46, 7.55) Idiopathic anaphylaxis 8,442 0.91 (0.70, 1. 18) Neuropathic pain 1.45 (1.01, 2.09) —--P' t' th ‘t‘ 4 0.38 -0.28 1.37 I 0.51, 3.67 I Retinopathy 019_m3.08 (0.76, 12.52) Rheumatoid arthritis 3, 093 -2 1. 27 (0. 89, 1. 84) Systemic lupus erythe 3 2.30 (1.47, 3.59) Systemic mastocytosis _ 0 6 4.79 (151,1518) Thrombo cerebrovasc accident 2 0.19 0.27 0.70 (0.17, 2.80) (CVA) In sum, an analysis of a large longitudinal claims database revealed that id diseases of SLE, neuropathic pain and systemic mastocytosis had a greater representation in HAE patients than other types of angioedema, suggesting that activity of the KKS may be contributing to the manifestations of these diseases.

Accordingly, patients having or at risk for HAE may be predisposed to KKS— associated es, including neuropathic pain, systemic lupus erythrmatosus, and ic mastocytosis. Thus, treatment with a pKal inhibitor, such as DX—2930 may reduce the risk for the development of such a KKS—associated disease.

Example 6: A Double-Blind Study including 3 Load Period and a Maintenance Period A randomized, double-blind, placebo-controlled, parallel arm study is carried out. Patients are selected for having HAE type I or II patients with at least 1 attack per 4 weeks. A run—in period is used to evaluate baseline HAE attack rate. The patients are subjected to a 1:1:1 randomization into 3 ent treatment arms (300 mg DX—2930, 150 mg DX—2930, or placebo) which are administered by subcutaneous injection. The study is designed with a load period and a maintenance period (Figure 14). Subjects are treated on Days 0, 7, and 14 during the loading period, followed by dosing every 2 weeks during the maintenance period (Days 28, 42, 56, 70, and 84).

OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each e disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or r purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an e of a generic series of equivalent or similar features.

From the above ption, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope f, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the .

EQUIVALENTS While several inventive ments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the on and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, als, and urations described herein are meant to be exemplary and that the actual parameters, dimensions, als, and/or urations will depend upon the speci?c ation or applications for which the ive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.

Inventive embodiments of the present disclosure 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, articles, materials, kits, and/or methods, if such features, systems, es, materials, kits, and/or s are not mutually inconsistent, is included within the inventive scope of the present sure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively t in other cases. Multiple elements listed with “and/or” should be ued in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be t other than the ts specifically identified by the “and/or” clause, whether related or unrelated to those ts specifically identified. Thus, as a non—limiting example, a reference to “A and/or B”, when used in conjunction with open—ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other ts); etc.

As used herein in the specification and in the claims, “or” should be tood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be reted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional ed items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating ive alternatives (i.e. “one or the other but not bot ”) when preceded by terms of exclusivity, such as “either,39 65 one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be tood to mean at least one element selected from any one or more of the ts in the list of elements, but not necessarily ing at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that ts may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non—limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally ing more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed 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 order in which the steps or acts of the method are d.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,a, cn‘including,’9 c4carrying,” “having,39 “containing,39 lving,” “holding,” “composed of,” and the like are to be understood to be open—ended, i.e., to mean including but not limited to. Only the transitional phrases sting of” and “consisting essentially of" shall be closed or semi—closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 21 1 1.03.

Claims (3)

WE CLAIM:

1. Use of an antibody in the manufacture of a medicament for preventing hereditary dema (HAE) attack or reducing the rate of HAE attack in a subject, wherein the antibody comprises a heavy chain (HC) complementarity determining region (CDR) 1 having the amino acid ce HYIMM (SEQ ID NO: 5), a HC CDR2 having the amino acid sequence GIYSSGGITVYADSVKG (SEQ ID NO: 6), a HC CDR3 having the amino acid sequence RRDEFDI (SEQ ID NO: 7), a light chain (LC) CDR1 having the amino acid sequence RASQSISSWLA (SEQ ID NO: 8), a LC CDR2 having the amino acid sequence KASTLES (SEQ ID NO: 9), and a LC CDR3 having the amino acid sequence QQYNTYWT (SEQ ID NO: 10), wherein the antibody is formulated in a pharmaceutically acceptable carrier comprising sodium phosphate at a tration of 30 mM, citric acid, histidine at a concentration of 50 mM, sodium chloride at a concentration of 90 mM, and polysorbate 80 at 0.01%, pH 6.0, wherein the medicament is to be administered at about 300 mg of the antibody every two to four weeks at least two times, and wherein the subject is a patient experiencing at least two HAE attacks per year prior to the administration.

2. The use according to claim 1, wherein the antibody comprises a HC variable domain having the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGI TVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWG QGTMVTVSS (SEQ ID NO: 3) and a light chain (LC) variable domain having the amino acid sequence SPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGV PSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK (SEQ ID NO:

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