Plasma kallikrein-binding protein in treatment of hereditary angioedema and use thereof

Abstract

To provide a method for treating hereditary angioedema.SOLUTION: The present invention provides a plasma kallikrein-binding protein such as an antibody binding to active plasma kallikrein in the treatment of hereditary angioedema, and a method for using the protein.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing dates of U.S. Provisional Application No. 61 / 929,716, filed January 21, 2014, U.S. Provisional Application No. 61 / 944,361, filed February 25, 2014, and U.S. Provisional Application No. 62 / 021,397, filed July 7, 2014, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Plasma kallikrein is a contact phase serine protease that is a potential drug target for different inflammatory, cardiovascular, infectious (sepsis) and oncological diseases (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). The contact phase is activated either by factor XII upon exposure to foreign or negatively charged surfaces or by prolylcarboxypeptidase on the endothelial cell surface (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation via feedback activity of factor XII and enhances inflammation via production of the proinflammatory nonapeptide bradykinin. As the major kininogenase in the circulation, plasma kallikrein plays a major role in the production of bradykinin in the vasculature. A genetic deficiency in the C1 inhibitor protein (C1-INH), the main natural inhibitor of plasma kallikrein, results in hereditary angioedema (HAE). HAE patients suffer from attacks of painful edema, often precipitated by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure is based in part on unexpected results from pharmacokinetic studies and pharmacokinetic modeling showing that a dose (e.g., about 100 mg to 300 mg) of an antibody that binds to the active form of human plasma kallikrein that maintains a plasma concentration of the antibody above 80 nM is sufficient to show a beneficial (e.g., prophylactic) effect in the treatment of hereditary angioedema. Furthermore, dosing DX-2930 in an amount of 100 mg every two weeks or in an amount of 300 mg every four weeks maintains a steady state plasma drug concentration above 80 nM, and dosing DX-2930 in an amount of 300 mg every two weeks maintains a steady state plasma drug concentration above 200 nM.

[0004] The present disclosure is also based in part on the unexpected discovery that an antibody that binds to the active form of human plasma kallikrein exhibited superior therapeutic efficacy in the treatment of hereditary angioedema (HAE) at various doses (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg) with no evidence of dose-limiting toxicity at single doses up to 3.0 mg / kg. Pharmacokinetic results showed that DX-2930 had linear dose-dependent exposure across dose groups and an average elimination half-life of 17-20 days after a single injection in healthy subjects. Pharmacodynamic results from two different exploratory biomarker assays confirmed ex vivo plasma kallikrein inhibition in a dose- and time-dependent manner.

[0005] Thus, one aspect of the disclosure features a method of treating HAE (e.g., Type I, II, or III) comprising administering to a subject in need thereof an antibody that binds an active form of pKal in an effective amount (e.g., 100 mg to 400 mg, or 100 to 300 mg) such that a plasma concentration of the antibody in the subject is greater than about 80 nM. In some embodiments, the antibody (e.g., DX-2930) is administered at 100 mg every two weeks. In some embodiments, the antibody (e.g., DX-2930) is administered at 300 mg every two weeks or every four weeks.

[0006] Another aspect of the disclosure features a method of treating HAE (e.g., Type I, II, or II), comprising: (a) administering to a subject in need thereof an antibody (e.g., a full-length antibody or an antigen-binding fragment thereof) that binds active plasma kallikrein in a first dose; (b) measuring a plasma concentration of the antibody in the subject; and (c) administering to the subject a second dose of the antibody if the plasma concentration of the antibody is less than about 80 nM. In some embodiments, the first dose, the second dose, or both, is between 100 mg and 400 mg or between 100 mg and 300 mg (e.g., 100 mg or 300 mg of DX-2930). In some embodiments, the second dose is greater than the first dose.

[0007] Thus, one aspect of the disclosure features a method of treating HAE (e.g., type I, II, or III) comprising administering a single dose of an isolated antibody to a subject in need thereof, where the antibody (e.g., a full-length antibody or an antigen-binding fragment thereof) binds active plasma kallikrein (e.g., does not bind prekallikrein). Optionally, the method further includes monitoring levels of creatine phosphokinase in the subject before and after treatment. In some embodiments, a single dose of any of the antibodies described herein is 0.1-3 mg / kg (e.g., 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg).

[0008] In another aspect, the disclosure provides a method of treating hereditary HAE, comprising administering to a subject in need thereof multiple doses of an isolated antibody (e.g., a full-length antibody or antigen-binding fragment thereof) that binds active plasma kallikrein (e.g., an antibody that binds human active plasma kallikrein but does not bind human prekallikrein), wherein each of two consecutive administrations is at least two weeks (e.g., three weeks, four weeks, five weeks, or six weeks) apart. In some embodiments, at least one dose of the multiple doses is 0.1-3 mg / kg. For example, each dose of the multiple doses is 3 mg / kg. In some embodiments, the antibody is administered monthly (e.g., every 28 days), for example, for six months.

[0009] In yet another aspect, the disclosure features a method of treating HAE, the method including: (i) administering to a subject in need thereof one or more doses of an isolated antibody (e.g., a full-length antibody or antigen-binding fragment thereof) that binds active plasma kallikrein (e.g., an antibody that binds human active plasma kallikrein but does not bind human prekallikrein); (ii) measuring the level of inhibition of plasma kallikrein by the antibody in the subject after the last administration; and (iii) if the level of inhibition is below the minimum therapeutic level, administering an additional dose of the antibody to the subject. In some embodiments, the one or more doses are 0.1-3 mg / kg (e.g., 0.1 mg / kg, 1 mg / kg, or 3 mg / kg for each dose). In one example, the minimum therapeutic level represents a serum or plasma concentration of the antibody of less than about 80 nM.

[0010] In any of the methods described herein, the anti-pallikrein antibody can be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, the antibody binds active plasma kallikrein and does not bind prekallikrein.

[0011] In some embodiments of any one of the methods described herein, the antibody binds to the same epitope as DX-2930 or competes with DX-2930 for binding to active plasma kallikrein. In some embodiments, the antibody comprises heavy chain CDRs identical to DX-2930, light chain CDRs identical to DX-2930, or both. In some embodiments, the antibody is DX-2930, a full-length IgG antibody described herein, or an antigen-binding fragment thereof.

[0012] In some embodiments of any one of the methods described herein, the antibody can be administered by subcutaneous administration. In some embodiments, the subject is a human patient suffering from, suspected of having, or at risk of having an attack of HAE. For example, the methods described herein are for the prophylactic treatment of HAE.

[0013] In some embodiments of any one of the methods described herein, the method further comprises monitoring the level of creatine phosphokinase in the subject before and after treatment or during the course of treatment. If an elevation in creatine phosphokinase is observed, the dose of the antibody (e.g., DX-2930) may be reduced or treatment may be terminated.

[0014] In yet another aspect, the disclosure provides a method for determining an optimal dose (e.g., optimal prophylactic dose) for treating hereditary angioedema (HAE) in a subject, comprising: (a) administering (e.g., subcutaneously) an initial dose of any of the antibodies described herein that bind active plasma kallikrein (e.g., DX-2930 or an antigen-binding fragment thereof) to a subject in need thereof; (b) measuring a plasma concentration of the antibody in the subject; and (c) increasing the dose of the antibody if the plasma concentration of the antibody is less than about 80 nM, wherein a dose that maintains a plasma concentration of the antibody above about 80 nM is selected as the optimal prophylactic dose for the subject. In some embodiments, the subject is a human patient who is not exhibiting symptoms of HAE at the time the antibody is administered. In some embodiments, the initial dose is about 100 mg to 400 mg or 100 mg to 300 mg (e.g., 100 mg or 300 mg of DX-2930).

[0015] The above method may further include monitoring the level of creatine phosphokinase in the subject before and after treatment, or during the course of treatment. Additionally, the method may further include reducing the dose of the antibody or terminating treatment if an elevation of creatine phosphokinase is observed.

[0016] In any of the methods described herein, the plasma concentration of antibody can be measured by an assay of plasma kallikrein activity or by an immunoassay.

[0017] Also within the scope of the present disclosure are (a) a pharmaceutical composition for use in treating HAE or determining optimal dosages of agents for treating HAE, the pharmaceutical composition comprising any of the anti-kallikrein antibodies described herein and a pharma- ceutical acceptable carrier, and (b) the use of the pharmaceutical composition to manufacture a medicament for the treatment of HAE. Use of the antibody for its intended purpose can be carried out under the administration conditions described herein.

[0018] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows the mean concentrations of DX-2930 following subcutaneous (SC) administration to healthy subjects. Concentration-time plots for each dose cohort are shown on a logarithmic scale. Error bars are standard deviation. The profile demonstrated linear, dose-dependent exposure. Parallel elimination phases across dose groups are consistent with a normally acting antibody with dose-independent kinetics where all doses act uniformly. [Diagram 2] FIG. 1 is a graph depicting predicted plasma concentrations following repeated dosing with DX-2930 at 3 mg / kg via subcutaneous administration every 28 days in healthy subjects. [Diagram 3] 1 is a graph depicting general principles of drug use for the prevention of disease states. [Figure 4] 1 is a graph depicting the general principles of use of DX-2930 for the prevention of HAE. [Diagram 5] 1 is a graph showing the comparative inhibitory activity of DX-2930 and ecallantide against pKal in vitro. [Figure 6] 1 is a graph depicting an alternative hypothesis regarding the conditions required to continuously maintain plasma concentrations of DX-2930 at or above 80 nM for the treatment and / or prevention of HAE. Alternatively, plasma concentrations of DX-2930 required for the treatment and / or prevention of HAE may be lower or higher than 80 nM. [Figure 7] FIG. 1 is a graph showing that plasma drug levels of 80 nM or greater are achieved following administration of a single 3 mg / kg dose of DX-2930. [Figure 8] 13 depicts pharmacokinetic (PK) modeling of chronic DX-2930 administration in healthy subjects, delivered subcutaneously at 3 mg / kg every 28 days. [Figure 9] 1 is a graph depicting the pharmacodynamic (PD) effects of DX-2930 at 3 mg / kg SC every 28 days in healthy subjects. [Figure 10] Graphs depicting pharmacodynamic (PD) and pharmacokinetic (PK) data following a single dose of DX-2930 (3 mg / kg). PD data are plotted as relative % inhibition of pKal activity over time. PK data are plotted as plasma DX-2930 concentration (nM) over time. *P-values ​​< 0.05 (Day 5, 3 mg / kg vs. placebo). [Figure 11] 1 is a graph depicting the pharmacodynamic (PD) activity of DX-2930 against natural biological substrates (HMWK cleavage was measured as the generation of two chains of HMWK). [Figure 12] 1 is a graph depicting sustained DX-2930 bioactivity over time following a single dose of 3 mg / kg DX-2930. *P value < 0.05 (3 mg / kg and day 28 vs. prophylactic dosing). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition Before further describing the present invention, for convenience, certain terms used in the specification, examples and appended claims are individually defined. Other terms are defined as they become apparent herein.

[0021] The singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0022] The term "antibody" refers to a protein that includes at least one immunoglobulin variable domain (Var region) or immunoglobulin variable domain (Var region) sequence. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH or HV), and a light (L) chain variable 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" includes antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39)) as well as complete antibodies. Antibodies may have structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes). Antibodies may be from any source, although primate (human and non-human primate) and primatized antibodies are preferred.

[0023] The VH and VL regions can be further divided into regions of hypervariability called "complementarity determining regions (CDRs)" interspersed with more conserved regions called "framework regions (FRs)". The extent of the framework regions and CDRs has been defined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The Kabat definition is used herein. Each VH and VL is generally composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0024] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence that is capable of forming the structure of an immunoglobulin variable domain such that one or more CDR regions are arranged in a conformation suitable for an antigen binding site. For example, the sequence may include all or a portion of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may exclude 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 modifications. In one embodiment, a polypeptide comprising an immunoglobulin variable domain sequence is capable of combining with another immunoglobulin variable domain sequence to form a structure that preferentially interacts with an antigen binding site, e.g., plasma kallikrein.

[0025] The VH or VL chain of the antibody can further comprise all or a portion of a heavy or light chain constant region to form a heavy or light chain immunoglobulin, respectively. In one embodiment, the antibody is a tetramer of two heavy and two light chain immunoglobulins, the heavy and light chain immunoglobulins being internally linked, for example, by disulfide bonds. In IgG, the heavy chain constant region comprises the three immunoglobulin domains CH1, CH2 and CH3. The light chain constant region comprises a CL domain. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. The constant region of the antibody generally mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first complement (Clq) of the classical complement pathway. The light chain of the immunoglobulin can be of type kappa or lambda. In one embodiment, the antibody is glycosylated. The antibody may be functional for antibody-dependent cytotoxicity and / or complement-mediated cytotoxicity.

[0026] One or more regions of the antibody may be human or effectively human. For example, one or more variable regions may be human or effectively human. For example, one or more CDRs may be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3. The CDRs of each light chain (LC) and / or heavy chain (HC) may be human. The HC CDR3 may be human. One or more framework regions may be human, e.g., FR1, FR2, FR3, and / or FR4 of the HC and / or LC. For example, the Fc region may be human. In one embodiment, all framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell or a non-hematopoietic cell that produces immunoglobulins. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, framework (FR) residues of a selected Fab can be converted to the amino acid species of the corresponding residue in the most similar primate germline gene, particularly in human germline genes. One or more constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of the immunoglobulin variable domain, constant region, and constant domains (CH1, CH2, CH3, and / or CL1), or the entire antibody, can be human or effectively human.

[0027] All or part of an antibody may be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as many immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 kDa or about 214 amino acids) are encoded by a variable region (about 110 amino acids) gene at the NH2-terminus 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 (about 116 amino acids) gene and one of the other above-mentioned constant region genes, such as gamma (encoding about 330 amino acids). The length of the human HC varies considerably, with the HC CDR3 varying from about 3 amino acid residues to more than 35 amino acid residues.

[0028] The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retain the property of specifically binding to a target of interest. Examples of binding fragments that are included in the term "antigen-binding fragment" of a full-length antibody and retain functionality include (i) Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, which is a bivalent fragment that contains two Fab fragments that are linked by disulfide bridges at the hinge region; (iii) Fd fragment, which is composed of VH and CH1 domains; (iv) Fv fragment, which is composed of the VL and VH domains of a single arm of an antibody; (v) dAb fragment, which is composed of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that can be made as a single protein chain, in which the pair of VL and VH regions forms a monovalent molecule known as a single chain Fv (scFv). See, e.g., U.S. Patent Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0029] Antibody fragments can be obtained using any suitable technique, including conventional techniques known to those of skill in the art. The term "monospecific antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular target, e.g., epitope. This term includes "monoclonal antibodies" or "monoclonal antibody compositions," as used herein, to refer to a preparation of antibodies or fragments thereof of single molecular composition, regardless of how the antibody was made.

[0030] An antibody is "germlined" by replacing one or more non-germline amino acids in the framework region with the germline amino acid of the corresponding antibody, so long as binding characteristics are substantially retained.

[0031] The inhibition constant (Ki) provides a measure of the potency of an inhibitor; this constant is the concentration of inhibitor required to reduce the enzyme activity by half and is independent of the enzyme or substrate concentration. The apparent Ki (K i,app ) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of an inhibitor (e.g., an inhibitory binding protein) on the extent of the reaction (e.g., enzyme activity), and the change in the pseudo-first-order rate constant as a function of inhibitor concentration is fitted to the yield of Morrison's equation (Equation 1) to obtain an estimate of the apparent Ki value. Ki is the K i,app It is obtained from the y-intercept extracted from linear regression analysis of a plot of versus substrate concentration.

number

[0032] As used herein, "binding affinity" refers to the apparent binding constant or K A Refers to. A is the dissociation constant (K D ) is the reciprocal of the . The binding antibody has, for example, a specific target molecule, e.g., plasma kallikrein, and a binding affinity of at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 and 10 11 M -1 A higher affinity binding antibody for a first target compared to a second target can have a binding affinity of K A (or a number K D ) higher than the K for binding of the first target A(or a smaller number K D ). In such cases, the binding antibody has specificity for a first target (e.g., a protein in a first conformation, or a mimetic thereof) compared to a second target (e.g., the same protein in a second conformation, or a mimetic thereof; or a second protein). The difference in binding affinity (e.g., with respect to specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 It may be double.

[0033] Binding affinity can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of bound and free binding protein as a function of the binding protein (or target) concentration. The concentration of bound binding protein ("Bound") is related to the concentration of free binding protein ("Free") and the concentration of binding sites for the binding protein on the target by the following equation:

number

[0034] However, the exact K A It is not necessary to make a decision on this matter, which may be the case AIt is sufficient to obtain a quantitative measure of affinity, determined using methods such as, for example, ELISA or FACS analysis, that is proportional to, and can then be used for comparison to obtain a quantitative measure of affinity, or an inference of affinity, by activity in a functional assay, e.g., an in vitro or in vivo assay, such as to determine whether a higher affinity is, for example, two-fold higher.

[0035] The term "binding antibody" (or "binding protein" as used interchangeably herein) refers to an antibody capable of interacting with a target molecule. This term is used interchangeably with "ligand." "Plasma kallikrein binding protein" refers to an antibody capable of interacting with (e.g., binding to) plasma kallikrein, and particularly includes antibodies that preferentially or specifically interact with and / or inhibit plasma kallikrein. An antibody inhibits plasma kallikrein if it reduces the activity of plasma kallikrein compared to the activity of plasma kallikrein in the absence of the antibody and under identical conditions.

[0036] A "conservative amino acid substitution" refers to an amino acid residue being replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined by those skilled in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, 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).

[0037] One or more framework and / or CDR amino acid residues of the binding protein may comprise one or more mutations (e.g., substitutions (e.g., conservative substitutions or replacements of non-essential amino acids), insertions, or deletions) compared to the binding proteins described herein. The plasma kallikrein binding protein may have mutations (e.g., substitutions (e.g., conservative substitutions or replacements of non-essential amino acids), insertions, or deletions) relative to the proteins described herein (e.g., at least 1, 2, 3, or 4, and / or fewer than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations), e.g., mutations that have no substantial effect on the function of the protein. The mutations may be present in the framework regions, CDRs, and / or constant regions. In some embodiments, the mutations are present in the framework regions. In some embodiments, the mutations are present in the CDRs. In some embodiments, the mutations are present in the constant regions. Whether a particular substitution will be tolerated, i.e., will not adversely affect biological properties such as binding activity, can be predicted, for example, by assessing whether the mutation is conservative, or by the methods of Bowie, et al. (1990) Science 247:1306-1310.

[0038] An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that contains a sufficient number of human framework amino acid positions such that it does not elicit an immunogenic response in normal humans. An "effectively human" antibody is an antibody that contains a sufficient number of human amino acid positions such that it does not elicit an immunogenic response in normal humans.

[0039] "Epitope" refers to a site on a target compound that is bound by a binding protein (e.g., an antibody, such as a Fab or full-length antibody). When the target compound is a protein, the site can be composed entirely of amino acid building blocks, composed entirely of chemical modifications of amino acids of the protein (e.g., glycosyl sites), or a combination thereof. Overlapping epitopes contain at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature.

[0040] A first binding antibody "binds to the same epitope" as a second binding antibody if the first binding antibody binds to the same site on the target compound as the second binding antibody or binds to a site that overlaps with the site bound by the second binding antibody (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap in terms of amino acid sequence or other molecular features (e.g., glycosyl, phosphate, or sulfate groups)).

[0041] A first binding antibody "competes for binding" with a second binding antibody if binding of the first binding antibody to the epitope of the first binding antibody reduces (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second binding antibody that binds to the epitope of the second binding antibody. This 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., binding of the first binding antibody to the epitope of the first binding antibody causes a conformational change in the target compound that reduces the propensity of the second binding antibody to bind to the epitope of the second binding antibody).

[0042] Calculation of "homology" or "sequence identity" between two sequences (these terms are used interchangeably herein) is performed as follows: The sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). This optimal alignment is determined as the best score using the GAP program of the GCG software package, using a Blosum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by an amino acid residue or nucleotide that is identical to the corresponding position in the second sequence, 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 two sequences is a function of the number of identical positions shared by the sequences.

[0043] In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably 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 reference sequence. For example, the reference sequence may be the length of an immunoglobulin variable domain sequence.

[0044] A "humanized" immunoglobulin variable region is an immunoglobulin variable region that has been modified to contain a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in normal humans. "Humanized" immunoglobulins are described, for example, in U.S. Patent No. 6,407,213 and U.S. Patent No. 5,693,762.

[0045] An "isolated" antibody refers to an antibody that is at least 90% removed from at least one component of the natural sample from which the isolated antibody is obtained. An antibody can be "at least" a particular degree of purity if the subject species or population of species is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure by weight.

[0046] A "patient," "subject," or "host" (the terms are used interchangeably) treated by the subject methods can mean either a human or non-human animal.

[0047] The terms "prekallikrein" and "pre-plasma kallikrein" are used interchangeably herein and refer to the zymogen form of active plasma kallikrein, also known as prekallikrein.

[0048] As used herein, "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 amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains, e.g., have conservative amino acid substitutions) to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have a similar activity, e.g., binding activity, binding preference, or biological activity (or encode proteins with similar activity). In the case of antibodies, the second antibody has the same specificity and at least 50%, at least 25%, or at least 10% of the affinity compared to the same antigen.

[0049] Sequences similar or homologous (e.g., having at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity may be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, plasma kallikrein binding antibodies may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the antibodies described herein. In some embodiments, a plasma kallikrein binding antibody may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC framework regions (e.g., HC and / or LC FR1, 2, 3, and / or 4) to an antibody described herein (e.g., DX-2930). In some embodiments, a plasma kallikrein binding antibody may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the constant regions (e.g., CH1, CH2, CH3, and / or CL1) to an antibody described herein (e.g., DX-2930).

[0050] Furthermore, substantial identity exists to a complementary strand when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions).The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially purified form.

[0051] Statistical significance can be determined by any method known to one of skill in the art. Exemplary statistical tests include the Student's t-test, the non-parametric Mann-Whitney U test, and the non-parametric Wilcoxon statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. A particular binding protein may, for example, exhibit differences in specificity or binding that are statistically significant (e.g., P value < 0.05 or 0.02). For example, the terms "induce," "inhibit," "enhance," "elevate," "increase," "decrease," and the like, which indicate a distinguishable qualitative or quantitative difference between two states, may refer to a difference, e.g., a statistically significant difference, between two states.

[0052] A "therapeutically effective dose" preferably modulates a measurable parameter, such as plasma kallikrein activity, by a statistically significant degree, or by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to untreated controls. The ability of a compound to modulate a measurable parameter, such as a disease-related parameter, can be evaluated in an animal model system that is predictive of efficacy in human disorders and conditions. Alternatively, this property of a composition can be evaluated by testing the ability of the compound to modulate the parameter in vitro.

[0053] The term "treating" as used herein refers to the application or administration of a composition containing one or more active agents to a subject having an allergic disease, a symptom of an allergic disease, or a predisposition to an allergic disease, for the purpose of curing, curing, mitigating, alleviating, altering, remedying, ameliorating, improving, or affecting the disease, the symptoms of the disease, or the predisposition to the disease. "Prophylate treatment," also known as "preventive treatment," refers to treatment intended to protect against or reduce the risk of a disease to which the patient has been or may be exposed.

[0054] The term "preventing" a disease in a subject refers to administering a pharmaceutical treatment to a subject, such as administering an agent such that at least one of the diseases is prevented, i.e., administered prior to clinical manifestation of an undesirable pathology (e.g., a disease or other undesirable condition in a host animal) such that the host does not develop the undesirable pathology. "Preventing" a disease may also refer to a "prophylactic" or "prophylactic treatment."

[0055] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, since 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.

[0056] Plasma kallikrein-binding antibodies The plasma kallikrein binding antibodies used in the methods described herein may be full length (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) or may comprise only an antigen-binding fragment (e.g., Fab, F(ab')2 or scFv fragment). The binding antibody may comprise two heavy chain immunoglobulins and two light chain immunoglobulins, or may be a single chain antibody. The plasma kallikrein binding antibody may be a recombinant protein, such as a humanized antibody, a CDR-grafted antibody, a chimeric antibody, a deimmunized antibody, or an in vitro generated antibody, and may optionally comprise a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein binding antibody is a monoclonal antibody.

[0057] 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 mouse kallikrein) and includes at least one immunoglobulin variable region. For example, the antibody includes a heavy chain (HC) immunoglobulin variable domain sequence and / or a light chain (LC) immunoglobulin variable domain sequence. In one embodiment, the antibody binds to and inhibits plasma kallikrein, e.g., human plasma kallikrein and / or mouse kallikrein.

[0058] The antibody may comprise one or more of the following features: (a) human CDRs or human framework regions; (b) the HC immunoglobulin variable domain sequence 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 the CDRs of the HC variable domains described herein; (c) the LC immunoglobulin variable domain sequence 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 the CDRs of the LC variable domains described herein. (d) 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 (e.g., in whole or in a framework region or CDR) to a LC variable domain described herein; (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 (e.g., in whole or in a framework region or CDR) to a LC variable domain described herein; (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 a primate framework region; (h) 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 the CDR1 of the HC variable domain described herein (e.g., in whole or in a framework region or CDR); (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 a primate framework region; (h) 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 the CDR1 of the HC variable domain described herein; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid and differs by no more than 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of the HC variable domain described herein; (j) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid and differs by no more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of the HC variable domain described herein;(k) the LC immunoglobulin variable domain sequence comprises a CDR1 that differs by at least one amino acid and not more than 2, 3, 4, or 5 amino acids from the CDR1 of the LC variable domain described herein; (l) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid and not more than 2, 3, or 4 amino acids from the CDR2 of the LC variable domain described herein; (m) the LC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid and not more than 2, 3, 4, or 5 amino acids from the CDR3 of the LC variable domain described herein. (n) the LC immunoglobulin variable domain sequence differs by at least one amino acid and by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the LC variable domains described herein (e.g., in the entire or framework regions or CDRs); and (o) the HC immunoglobulin variable domain sequence differs by at least one amino acid and by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the HC variable domains described herein (e.g., in the entire or framework regions or CDRs).

[0059] The plasma kallikrein 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 kallikrein binding antibody or composition thereof is isolated from antibody cleavage fragments (e.g., DX-2930) that are inactive or partially active (e.g., binds plasma kallikrein with a Ki,app of 5000 nM or more) compared to the plasma kallikrein binding antibody. For example, the plasma kallikrein binding antibody is at least 70% free of such antibody cleavage fragments, and in other embodiments, is at least 80%, at least 90%, at least 95%, at least 99% or even 100% free of inactive or partially active antibody cleavage fragments.

[0060] The plasma kallikrein binding antibody may further inhibit plasma kallikrein, eg, human plasma kallikrein.

[0061] In some embodiments, the plasma kallikrein binding antibody does not bind prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein) but binds to the active form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein).

[0062] In certain embodiments, the antibody binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds an epitope that overlaps with the active site of plasma kallikrein.

[0063] In some embodiments, the antibody binds the same epitope or competes for binding with an antibody described herein.

[0064] The antibody should be at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , and 10 11 M -1 In one embodiment, the antibody can bind to plasma kallikrein, e.g., human plasma kallikrein, with a binding affinity of 1×10 -3 , 5×10 -4 s -1 , or 1 × 10 -4 s -1 Slower K off In one embodiment, the antibody binds to human plasma kallikrein at a concentration of 1×10 2 , 1×10 3 , or 5 × 10 3 M -1 s -1 Faster K onIn one embodiment, the antibody binds to plasma kallikrein but does not bind to tissue kallikrein and / or plasma prekallikrein as effectively as it binds to plasma kallikrein (e.g., the antibody binds to tissue kallikrein and / or plasma prekallikrein with a weak effect (e.g., 5-fold, 10-fold, 50-fold, or 100-fold less than a negative control, or no effect at all).

[0065] In one embodiment, the antibody is -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , and 10 -10 The antibodies inhibit human plasma kallikrein activity with a Ki of less than 1 M. The antibodies may have an IC of less than 100 nM, 10 nM, 1, 0.5, or 0.2 nM, for example. 50 For example, the antibody may modulate the activity of plasma kallikrein and the production of factor XIIa (e.g., from factor XII) and / or bradykinin (e.g., from high molecular weight kininogen (HMWK)). The antibody may inhibit the activity of plasma kallikrein and / or the production of factor XIIa (e.g., from factor XII) and / or bradykinin (e.g., from high molecular weight kininogen (HMWK)). The affinity of the antibody for human plasma kallikrein may be less than 100 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. D In one embodiment, the antibody inhibits plasma kallikrein but does not inhibit tissue kallikrein to the same extent that it inhibits plasma kallikrein (e.g., the antibody inhibits tissue kallikrein with a weak effect (e.g., 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold less than a negative control, or no effect at all).

[0066] In some embodiments, the antibodies have an apparent inhibition constant (K i,app ).

[0067] Plasma kallikrein binding antibodies may have their HC and LC variable domain sequences contained in a single polypeptide (eg, scFv) or on different polypeptides (eg, IgG or Fab).

[0068] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the antibody is an IgG, such as, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may be a soluble Fab. In other implementations, the antibody comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen binding 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, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable constructs.

[0069] In one embodiment, the antibody is a human or humanized antibody, or is non-immunogenic in humans. For example, the antibody comprises one or more human antibody 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 a human framework region. In one embodiment, the antibody comprises a human Fc domain, or an Fc domain at least 95, 96, 97, 98, or 99% identical to a human Fc domain.

[0070] In one embodiment, the antibody is a primate antibody or primatized antibody, or is non-immunogenic in humans. For example, the antibody comprises one or more primate framework regions, such as all primate framework regions, or framework regions that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to a primate framework region. In one embodiment, the antibody comprises a primate Fc domain, or an Fc domain that is at least 95%, 96%, 97%, 98%, or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscu (bonobo)), gorillas (Gorilla gorilla), gibbons (gibons), monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0071] In some embodiments, the affinity of the primate antibody for human plasma kallikrein is less than 1000 nM, less than 500 nM, less than 100 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. D , for example less than 10 nM, less than 1 nM, or less than 0.5 nM.

[0072] In certain embodiments, the antibody does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0073] In some embodiments, the antibody used in the methods described herein may be DX-2930 or a functional variant thereof as described herein, or may be an antibody that binds the same epitope as DX2930 or competes with DX-2930 for binding to active plasma kallikrein.

[0074] In one example, a functional variant of DX-2930 contains the same complementarity determining regions (CDRs) as DX-2930. In another example, a functional variant of DX-2930 contains the same VDRs as DX-2930. H and VL Compared to the FR of V H or V L The functional variants may contain one or more mutations (e.g., conservative substitutions) in any of the FRs of DX-2930. Preferably, such mutations do not occur at residues predicted to interact with one or more CDRs, which can be determined by routine techniques. In other embodiments, the functional variants described herein contain one or more mutations (e.g., 1, 2, or 3) within one or more CDR regions of DX-2930. Preferably, such functional variants retain the same regions / residues as the parent that contribute to antigen binding. In yet other embodiments, the functional variants of DX-2930 contain one or more mutations (e.g., 1, 2, or 3) within one or more CDR regions of DX-2930. H V that contains an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to H Chain and / or DX-2930 V L V has an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to L These variants can bind to the active form of plasma kallikrein and preferably do not bind to prekallikrein.

[0075] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, 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, et 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 a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0076] Preparation of antibodies Antibodies capable of binding PKal as described herein can be produced by any method known to those skilled in the art, see, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0077] In some embodiments, antibodies specific to a target antigen (e.g., human PKal or its catalytic domain) can be produced by conventional hybridoma technology. A full-length target antigen or a fragment thereof, optionally conjugated to a carrier protein such as KLH, can be used to immunize a host animal to produce antibodies that bind to the antigen. The route and schedule of immunization of the host animal will generally follow established conventional techniques for stimulating and producing antibodies, as further described herein. General techniques for the production of mouse antibodies, humanized antibodies, and human antibodies are known to those of skill in the art and are described herein. Any mammalian subject, including humans, or antibody producing cells therefrom, can be engineered to act as the basis for the production of mammalian cell lines, including human hybridoma cell lines. Generally, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, plantarly, and / or intradermally with an amount of an immunogen, including those described herein.

[0078] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or as modified by Buck, DW, et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used for hybridization. In general, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Any of the media described herein, supplemented or not with serum, can be used to culture hybridomas secreting monoclonal antibodies. As an alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce anti-PKal monoclonal antibodies as described herein. Hybridomas are expanded and subcloned as needed, and supernatants are assayed for anti-immunogen activity by conventional immunoassay techniques (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0079] Hybridomas that may be used as a source of antibodies include all derivatives and progeny cells of the parent hybridoma that produce monoclonal antibodies capable of interfering with the activity of PKal. Hybridomas producing such antibodies may be grown in vitro or in vivo using known techniques. Monoclonal antibodies may be isolated from the culture medium or body fluids, if desired, by conventional immunoglobulin purification steps such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If undesirable activity is present, it can be removed, for example, by passing the preparation through an adsorbent made from the immunogen bound to a solid phase, eluting or releasing the desired antibody from the immunogen. A population of antibodies (e.g., monoclonal antibodies) can be recovered by immunization of a host animal with the target antigen or a fragment containing the target amino acid sequence coupled to a protein that is immunogenic in the species to be immunized, such as, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalizing or derivatizing agent, such as maleimidobenzoylsulfonsuccinimide ester (linked through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR (where R and R1 are different alkyl groups).

[0080] If necessary, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in a vector in a host cell, which may then be expanded and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic engineering 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 be more similar to human constant regions to avoid immune responses when the antibody is used in human clinical trials and treatments. It may be preferable to genetically engineer the sequence of the antibody to obtain higher affinity for the target antigen and higher efficacy in terms of inhibiting the activity of PKal. It is clear to those skilled in the art that one or more polynucleotides can be altered to an antibody and still maintain its binding specificity for the target antigen.

[0081] In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic mice, which are designed to generate more desirable (e.g. fully human antibodies) or stronger immune responses, may also be used to generate humanized or human antibodies. Examples of such technology are Xenomouse® from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies may be recombinantly generated by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0082] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods, for example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment.

[0083] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into one or more expression vectors and then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. See, for example, International Patent Publication No. WO 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence with human heavy and light chain constant domains in place of the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, which have the binding specificity of a target antigen can be prepared.

[0084] Techniques developed for the production of "chimeric antibodies" are well known to those of skill in the art (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452).

[0085] Methods for constructing humanized antibodies are also well known to those skilled in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the VH and VL variable regions of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known to those skilled in the art. The framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences homologous to the parent non-human antibody are identified from any antibody gene database using the parent VH and Vl sequences as search queries. Human VH and VL acceptor genes are then selected.

[0086] The CDR regions in the selected human acceptor gene can be replaced with CDR regions from the parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain predicted to be important for interactions with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene.

[0087] Single chain antibodies can be prepared via recombinant techniques by combining nucleotide sequences encoding heavy chain variable regions and nucleotide sequences encoding light chain variable regions. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for producing single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce phage or yeast scFv libraries, and PKal-specific scFv clones can be identified from the libraries by routine methods. Positive clones can be further screened to identify those that inhibit PKal activity.

[0088] The antibodies obtained according to the methods known to those skilled in the art and described herein can be characterized using methods well known to those skilled in the art. For example, one method is to identify the epitope to which the antibody binds, or "epitope mapping". There are many methods known to those skilled in the art to map and characterize the location of an epitope on a protein, including analyzing the crystal structure of an antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by three-dimensional interactions of amino acids, which may not necessarily be contained in a single stretch (linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding with the antibody. Gene fragment expression assays involve fragmenting the open reading frame encoding the target antigen randomly or by specific genetic construction and determining the reactivity of the expressed fragment of the antigen with the antibody being tested. Gene fragments may be produced, for example, by PCR in the presence of radioactive amino acids and then transcribed and translated into protein in vitro. Binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles.Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In a further example, mutagenesis of the antigen-binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues that are required, sufficient and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the PKal polypeptide are replaced (swapped) with sequences from closely related but antigenically distinct proteins (such as other members of the neurotrophin protein family). The importance of a particular antigen fragment to the binding of the antibody can be evaluated by assessing the binding of the antibody to mutant PKals (e.g., the mutants described in Example 2 below).

[0089] Alternatively, to determine whether an antibody binds to the same epitope as another antibody, a competition assay can be performed using another antibody known to bind to the same antigen. Competition assays are well known to those of skill in the art.

[0090] For example, any suitable method known to those skilled in the art, such as the epitope mapping method described herein, can be used to determine whether an anti-PKal antibody binds one or more of the specific residues / segments in PKal described herein. Furthermore, the interaction of an antibody with one or more of the defined residues in PKal can be determined by routine techniques. For example, a crystal structure can be determined according to the method disclosed in Example 1 below, and thus the distance between a residue in PKal and one or more residues in an antibody can be determined. Based on such distance, it can be determined whether a particular residue in PKal interacts with one or more residues in an antibody. Furthermore, suitable methods, such as competitive assays and targeted mutation assays, can be used to determine the preferential binding of a candidate anti-PKal antibody to PKal compared to another target, such as a mutant PKal.

[0091] Alternatively, anti-PKal antibodies can be identified from antibody libraries, such as display libraries, according to methods known to those of skill in the art.

[0092] After an anti-PKal antibody has been identified using any method known to one of skill in the art and confirmed to be suitable for use in the treatments described herein, such an antibody may be produced by standard recombinant nucleic acid methods.

[0093] Generally, a nucleic acid sequence encoding a protein is cloned into a nucleic acid expression vector. Of course, if the protein comprises multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, and expressed in the same or different cells.

[0094] For example, some antibodies, such as Fabs, can be produced in bacterial cells, such as E. coli cells (see, e.g., Nadkarni, A. et al., 2007 Protein Expr Purif 52(1):219-29). For example, if the Fab is encoded by a sequence in a phage display vector that contains a suppressible stop codon between the display entity and the bacteriophage protein (or a fragment thereof), the nucleic acid of the vector can be introduced into a bacterial cell that cannot suppress the stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and / or medium.

[0095] Antibodies can also be produced in eukaryotic cells. In one embodiment, antibodies (e.g., scFv's) are expressed in yeast cells 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 20(3):291-300), Hanseula, or Saccharomyces.The skilled artisan will be familiar with the various aspects of the present invention, including, for example, oxygen conditions (see, e.g., Baumann K., et al. 2010 BMC Syst. Biol. 4:141), osmolality (see, e.g., Dragosits, M. et al., 2010 BMC Genomics 11:207), temperature (see, e.g., Dragosits, M. et al., 2009 J Proteome Res. 8(3):1380-92), fermentation conditions (see, e.g., Ning, D. et al. 2005 J. Biochem. and Mol. Biol. 38(3): 294-299), yeast strains (see, e.g., Kozyr, AV et al. 2004 Mol Biol (Mosk) 38(6):1067-75; Horwitz, AH. et al., 1988 Proc Natl Acad Sci USA 85(22):8678-82; Antibody production in yeast can be optimized by optimizing the culture medium with respect to the following: Bowdish, K. et al. 1991 J Biol Chem 266(18):11901-8), overexpression of proteins to enhance antibody production (see, e.g., Gasser, B. et al., 2006 Biotechol. Bioeng. 94(2):353-61), acidity level of the culture (see, e.g., Kobayashi H., et al., 1997 FEMS Microbiol Lett 152(2):235-42), and concentrations of substances and / or ions (see, e.g., Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):41-8). Furthermore, the yeast system can be used to produce antibodies with extended half-lives (see, e.g., Smith, BJ. et al. 2001 Bioconjug Chem 12(5):750-756).

[0096] In one preferred embodiment, the antibody is produced in mammalian cells. Preferred mammalian host cells for expressing cloned antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, and used with DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(1-2):65-80), and cells from transgenic animals, e.g., from transgenic mammals. For example, the cells are mammalian epithelial cells.

[0097] In some embodiments, plasma kallikrein binding antibodies are produced in plants or cell-free systems (see, eg, Galeffi, P., et al., 2006 J Transl Med 4:39).

[0098] In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vector may contain additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. 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 methotrexate, on a host cell into which the vector has been introduced. A preferred selectable marker gene is the dihydrofolate reductase (DHFR) gene (dhfr for methotrexate selection / amplification). - The vector contains the p53 gene (for use in a host cell) and the neo gene (for G418 selection).

[0099] In an exemplary system for recombinant expression of an antibody, or antigen binding protein thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is transfected into the dhfr vector by calcium phosphate mediated transfection. - The antibody heavy and light chain genes are introduced into CHO cells. In the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to an enhancer / promoter control (e.g., CMV enhancer / AdMLP promoter control or SV40 enhancer / AdMLP promoter control, such as from SV40, CMV, adenovirus, etc.) to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography on a protein A or protein G binding matrix.

[0100] 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 an IgG molecule is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site of modification with biantennary oligosaccharides. This glycosylation has been shown to be necessary for effector functions mediated by Fcg receptors and complement C1q (Burton and Woof, 1992, Adv. Immunol. 51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In one embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain also includes other eukaryotic post-translational modifications.

[0101] Antibodies can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that contains a milk-specific promoter, a nucleic acid encoding the antibody of interest, and a signal sequence for secretion. Milk produced by females of such transgenic mammals contains the antibody of interest secreted therein. The antibody can be purified from the milk or used directly for several applications.

[0102] Pharmaceutical Compositions One or more antibodies described herein may be present in a composition, such as, for example, a pharma- ceutically acceptable composition or pharmaceutical composition. The plasma kallikrein binding antibody can be formulated with a pharma- ceutically acceptable carrier. In some embodiments, 100 mg to 300 mg of an antibody described herein (e.g., a dose of 100 mg or 300 mg of DX-2930) is present in a composition, such as, for example, a pharma- ceutically acceptable composition or pharmaceutical composition, optionally including a pharma- ceutically acceptable carrier.

[0103] Pharmaceutically acceptable carriers include any and all pharmacologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion), although carriers suitable for inhaled and intranasal administration are also contemplated.

[0104] Pharmaceutically acceptable salts are salts that retain the desired biological activity of a compound and do not impart undesired toxicological effects (see, e.g., Berge, SM, 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 salts derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and salts derived from non-toxic organic acids, such as fatty mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, fatty and aromatic sulfonic acids, and the like. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, and salts derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0105] The compositions may be in various forms. These include liquid, semi-solid and solid dosage forms, such as, for example, solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form may depend on the intended mode of administration and therapeutic application. Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for human administration with antibodies. Exemplary modes of administration are 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 intraperitoneal injection.

[0106] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0107] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other designated structure suitable for high concentration of the drug. Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in a suitable solvent containing one or a combination of the above-mentioned ingredients, and then sterile filtering as necessary. In general, dispersions can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying to collect the powder of the active ingredient and any additional desired ingredients from their previously sterile-filtered solutions. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0108] Plasma kallikrein binding antibodies can be administered by a variety of methods, including intravenous injection or infusion. For example, for some therapeutic uses, plasma kallikrein binding protein is administered at a dose of about 1-100 mg / m 2 or 7 to 25 mg / m 2The active compound may be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg / min to reach a dose of 100 mg / min. The route and / or form of administration varies depending on the desired results. In certain embodiments, the active compound may be prepared with a carrier that protects the compound from rapid release, such as a sustained release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods are available for the preparation of such formulations. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., 1978, Marcel Dekker, Inc., New York.

[0109] The pharmaceutical composition can be administered using a medical device. For example, in one embodiment, the pharmaceutical compositions disclosed herein can be administered using a device such as, for example, a needleless hypodermic injection device, a pump, or an implant.

[0110] In certain embodiments, plasma kallikrein binding proteins can be formulated to maintain proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if necessary), these compounds can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, for example, VV Ranade, 1989, J. Clin. Pharmacol. 29:685).

[0111] The dosage regimen is adjusted to provide the optimum desired response (e.g., 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 requirements of the therapeutic situation. In particular, it is advantageous to formulate parenteral compositions in unit dosage forms for ease of administration and uniformity of dosage. Unit dosage forms as described herein refer to physically discrete units suitable as unitary doses for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms are determined by and may depend directly on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the individual sensitivity limits inherent in the technology of compounding such active compounds for treatment.

[0112] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the antibodies described herein is 0.1-20 mg / kg, more preferably 1-10 mg / kg. Anti-plasma kallikrein antibodies are preferably 1-100 mg / kg. 2 or about 5 to 30 mg / m 2 The composition can be administered, for example, by intravenous infusion, at a rate of, for example, less than 30, 20, 10, 5, or 1 mg / min to reach a dose of 100 mg / min. Dosage values ​​can vary with the type and severity of the condition to be alleviated. For a particular subject, a particular dosage regimen can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition.

[0113] In some embodiments, a therapeutically or prophylactically effective amount of an antibody described herein (e.g., DX-2930) is from 30 to 400 mg, 30 to 300 mg, 30 to 250 mg, 30 to 200 mg, 30 to 150 mg, 30 to 100 mg, 30 to 50 mg, 50 to 400 mg, 50 to 300 mg, 50 to 250 mg, 50 to 200 mg, 50 to 150 mg, 50 to 100 mg, 100 mg, 200 mg, 25 ... The effective amount of the therapeutically or prophylactically effective amount is 00-400 mg, 100-300 mg, 100-250 mg, 100-200 mg, 100-150 mg, 150-400 mg, 150-300 mg, 150-250 mg, 150-200 mg, 200-400 mg, 200-300 mg, 200-250 mg, 250-400 mg, 250-300 mg, or 300-400 mg, or any integer therebetween. In some embodiments, the therapeutically or prophylactically effective amount is 30-300 mg. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg or more. In some embodiments, the therapeutically or prophylactically effective amount is 400 mg or more. In some embodiments, the therapeutically or prophylactically effective amount is 100-300 mg (100 mg, 150 mg, 200 mg, 250 mg, or 300 mg).

[0114] In some embodiments, a therapeutically or prophylactically effective amount of an antibody described herein (e.g., DX-2930) is 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, or 400 mg. In some embodiments, a therapeutically or prophylactically effective amount is 30 mg, 100 mg, or 300 mg. In some embodiments, the therapeutically or prophylactically effective amount is 100 mg or 300 mg. In some embodiments, the therapeutically or prophylactically effective amount is 100 mg. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg.

[0115] In some embodiments, the therapeutically or prophylactically effective amount is administered at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more. In some embodiments, the therapeutically or prophylactically effective amount is administered daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or more. In some embodiments, the therapeutically or prophylactically effective amount is 100 mg or 300 mg, and the amount is administered every 2 weeks or every 4 weeks. In some embodiments, the therapeutically or prophylactically effective amount is 100 mg, and the amount of the antibody is administered every 2 weeks. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg, and the amount of the antibody is administered every 2 weeks or every 4 weeks. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg, and the amount of the antibody is administered every 2 weeks. In some embodiments, the therapeutically or prophylactically effective amount is 300 mg, which is administered every four weeks.

[0116] In some embodiments, a therapeutically or prophylactically effective amount is an amount that maintains a plasma or serum concentration of the antibody at greater than about 80 nM (e.g., greater than 100 nM, greater than 150 nM, or about 200 nM). In some embodiments, the amount of antibody is effective to maintain a plasma or serum concentration of the antibody in the range of about 80-300 nM, e.g., 80-100 nM, 80-120 nM, 80-150 nM, 100-150 nM, 100-200 nM, 150-200 nM, or 200-300 nM. For example, plasma or serum concentrations can be measured using a suitable assay, such as a plasma kallikrein assay, such as the assays described herein, an immuno-based assay, such as an ELISA assay or Western blot assay for determining cleaved kininogen, as described herein, or by mass spectrometry.

[0117] The pharmaceutical compositions disclosed herein may include a "therapeutically effective amount" or a "prophylactically effective amount" of the plasma kallikrein binding proteins disclosed herein.

[0118] kit One or more of the plasma kallikrein binding antibodies described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) a plasma kallikrein binding antibody, e.g., a composition (e.g., a pharmaceutical composition) comprising the plasma kallikrein binding antibody, and, optionally, (b) informational material. The informational material can be descriptive, instructional, commercially available, or other material related to the methods described herein and / or the use of the plasma kallikrein binding antibody, e.g., the plasma kallikrein binding antibody for the methods described herein. In some embodiments, the kit includes one or more doses of the plasma kallikrein binding antibody, e.g., DX-2930. In some embodiments, the one or more doses are 100 mg or 300 mg.

[0119] The informational material of the kit is not limited in this form. In one embodiment, the informational material may include information about the production of the compound, the molecular weight of the compound, concentration, expiration date, batch or production site information, etc. In one embodiment, the informational material relates to using the antibody to treat, prevent, or diagnose disorders and conditions, e.g., diseases or conditions associated with plasma kallikrein.

[0120] In one embodiment, the informational material may include instructions for administering the plasma kallikrein binding antibody in a suitable manner for carrying out the methods described herein, e.g., in an appropriate dose, dosage form, administration format, or administration schedule (e.g., a dose, dosage form, administration schedule, or administration format described herein). In another embodiment, the informational material may include instructions for administering the plasma kallikrein binding antibody to a suitable subject, e.g., a human having or at risk for a plasma kallikrein-associated disease or condition. For example, the material may include instructions for administering the plasma kallikrein binding protein to a patient having a disorder or condition described herein, e.g., a plasma kallikrein-associated disease, e.g., according to an administration schedule described herein. The informational material of the kit is not limited in this form. Often, the informational material, e.g., instructions, is provided in printed form, but may be in other formats, such as a computer readable medium.

[0121] The plasma kallikrein binding antibody can be provided in any form, for example in liquid, dried, or lyophilized form. The plasma kallikrein binding antibody is preferably substantially pure and / or sterile. When the plasma kallikrein binding antibody is provided in a liquid formulation, the liquid formulation is preferably an aqueous solution, and a sterile liquid formulation is preferred. When the plasma kallikrein binding antibody is provided in a dry form, it is generally reconstituted by adding a suitable solvent. A solvent, such as, for example, sterile water or a buffer, can optionally be provided in the kit.

[0122] The kit may include one or more containers for the composition comprising the plasma kallikrein binding antibody. In some embodiments, the kit includes separate containers, dividers or compartments for the composition and the informational material. For example, the composition may be included in a bottle, vial, or syringe, and the informational material may be included in association with the container. In other embodiments, the separate components of the kit are included in a single undivided container. For example, the composition may be included in a bottle, vial, or syringe that has attached thereto informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each including one or more unit dosage forms (e.g., dosage forms described herein) of the plasma kallikrein binding antibody. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each including a single unit dose of the plasma kallikrein binding antibody. The containers of the kit may be airtight, waterproof (e.g., impervious to changes in moisture or evaporation), and / or light-tight.

[0123] The kit optionally includes a device suitable for administration of the composition, such as, for example, a syringe, or any such delivery device. In one embodiment, the device is an implantable device that dispenses a metered dose of the antibody. The disclosure also features methods of providing the kit, for example, by incorporating the components described herein.

[0124] treatment In some aspects, the disclosure provides for the use of antibodies that bind to active plasma kallikrein (e.g., human active plasma kallikrein) in treating HAE. Antibodies suitable for use in the treatments described herein include DX-2930 or functional variants thereof described herein, antibodies that bind the same epitope as DX-2930, or antibodies that compete with DX-2930 for binding to human active plasma kallikrein.

[0125] Hereditary angioedema Hereditary angioedema (HAE), also known as "Quincke's edema", C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent episodes of severe swelling (edema), which can affect, for example, the limbs, face, genitals, digestive tract, and airways. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction, which can include throat swelling and sudden hoarseness; recurrent episodes of abnormal abdominal cramps with no clear cause; and / or intestinal swelling, which can be severe and cause abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with this HAE develop a non-itchy rash called erythema marginatum during attacks.

[0126] Airway swelling can be life-threatening and some patients die. Mortality is estimated at 15-33%. HAE results in approximately 15,000-30,000 emergency department visits per year.

[0127] Trauma or stress, such as dental procedures, illness (e.g., viral illnesses such as colds and flu), menstruation, and surgery, can trigger attacks of edema. To prevent acute attacks of HAE, patients can try to avoid certain stimuli that have previously caused attacks. However, in many cases, attacks occur without a known trigger. Generally, symptoms of HAE first appear in children and worsen during adolescence. On average, untreated individuals experience attacks every 1-2 weeks, with most episodes lasting about 3-4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, and even among people within the same family.

[0128] There are three types of HAE, known as types I, II, and III, all of which can be treated by the methods described herein. HAE is estimated to affect 1 in 50,000 people, with type I accounting for approximately 80% of HAE cases, type II accounting for approximately 15% of HAE cases, and type III being very rare. Type III is the most recently described form and was originally thought to occur only in women, although families in which males are affected have been identified.

[0129] HAE is inherited in an autosomal dominant pattern, whereby an affected person may inherit a mutation from one affected parent. De novo mutations in genes can also occur, so HAE can occur in people with no family history of the disease. It is estimated that 20-25% of cases result from de novo spontaneous mutations.

[0130] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene provides instructions for making the C1 inhibitor protein, which is important in controlling inflammation. C1 inhibitor inhibits the activity of certain proteins that promote inflammation. Mutations that cause hereditary angioedema type I reduce the levels of C1 inhibitor in the blood. In contrast, mutations that result in type II result in the production of an abnormally functioning C1 inhibitor. Without adequate levels of functional C1 inhibitor, excessive amounts of bradykinin are produced. Bradykinin promotes inflammation by increasing the leakage of fluid through blood vessel walls into the body's tissues. The accumulation of excess fluid in the body's tissues causes the development of swelling seen in individuals with hereditary angioedema types I and II.

[0131] Mutations in the F12 gene have been associated with some cases of hereditary angioedema type III. The F12 gene provides the instructions for making clotting factor XII. In addition to playing a key role in blood clotting (clotting), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Certain mutations in F12 lead to the production of factor XII with increased activity. As a result, more bradykinin is produced and blood vessel walls become leakier, which leads to the development of swelling. The cause of other cases of hereditary angioedema type III is still unknown. Mutations in one or more as yet unidentified genes may be responsible for the disorder in these cases.

[0132] Although HAE may present similarly to other forms of edema due to allergies or other medical conditions, it has significantly different causes and treatments. When hereditary angioedema is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are generally ineffective in HAE. However, epinephrine may be used in some cases for life-threatening reactions. Misdiagnosis has also led to unnecessary exploratory surgery in patients with abdominal swelling, and abdominal pain has been inappropriately diagnosed as psychosomatic in some HAE patients.

[0133] C1 inhibitor treatment, as well as other treatments for HAE, are described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0134] Acute treatment of an HAE attack is provided to stop the progression of edema as soon as possible. Concentration of C1 inhibitor from donor blood administered intravenously is one acute treatment. However, this treatment is not available in many countries. In emergency situations where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP), which also contains C1 inhibitor, can be used as an alternative.

[0135] C1 inhibitor purified from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are currently available in the United States, and two C1 inhibitor products are currently available in Canada. Berinert P (CSL Behring) is pasteurized and was approved by the FDA in 2009 for acute attacks. Cinryze (ViroPharma) is nanofiltered and was approved by the FDA in 2008 for prophylaxis. Rhucin (Pharming) is an investigational recombinant C1 inhibitor that does not carry the risk of transmission of infectious diseases from human blood-borne pathogens.

[0136] Treatment of acute HAE attacks may also include medication for pain relief and / or intravenous fluids.

[0137] Other modalities of treatment can stimulate the synthesis of C1 inhibitor or reduce the consumption of C1 inhibitor. Androgen medications such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0138] Helicobacter pylori may trigger abdominal attacks. Antibiotics to treat Helicobacter pylori reduce abdominal attacks.

[0139] Newer treatments attack the contact cascade: ecallantide (KALBITOR®, DX-88, Dyax) inhibits plasma kallikrein and is approved in the United States; icatibant (FIRAZYR®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0140] Diagnosis of HAE may depend, for example, on family history and blood tests. Research findings related to HAE types I, II, and III are described, for example, in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, C1 inhibitor levels are decreased, C4 levels are also decreased, but C1q levels are normal. In type II HAE, C1 inhibitor levels are normal or increased, but C1 inhibitor function is abnormal. C4 levels are decreased, and C1q levels are normal. In type III, C1 inhibitor, C4, and C1q levels are all normal.

[0141] For example, symptoms of HAE can be assessed using questionnaires, such as questionnaires completed by the patient, clinician, or family member. Such questionnaires are known to those of skill in the art and include, for example, VAS (visual analog scale). See, e.g., McMillan, CV et al. Patient. 2012;5(2):113-26.

[0142] Treatment of HAE with anti-PKal antibodies The present disclosure provides methods of treating hereditary angioedema (HAE) (e.g., alleviating, stabilizing, or eliminating one or more symptoms thereof) by administering 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 described herein. Additionally, methods of treating HAE by administering an antibody described herein (e.g., a therapeutically effective amount of an antibody described herein), e.g., according to a dosing schedule described herein or in combination with a second treatment, e.g., one other agent described herein. The present disclosure also provides methods of preventing HAE or symptoms thereof by administering an antibody described herein (e.g., a prophylactically effective amount of an antibody described herein) to a subject at risk of developing HAE (e.g., a subject who has a family member with HAE or a subject who is genetically predisposed to HAE), e.g., according to a dosing schedule described herein. In some examples, the subject may be a human patient who does not have symptoms of HAE at the time of treatment.

[0143] Antibodies that bind to plasma kallikrein, for example as described herein, have therapeutic and prophylactic utility, particularly in human subjects. These antibodies are administered to subjects, or even to cells in culture, for example in vitro or ex vivo, to treat, prevent, and / or diagnose various disorders and conditions, including, for example, plasma kallikrein-associated diseases. For example, these binding proteins can be used to modify the effect of plasma kallikrein released from cells in culture (Lilla et al., J. Biol Chem. 284(20):13792-13803(2009)). Treatment includes administering an amount effective to alleviate, ameliorate, alter, treat, ameliorate, improve, or affect the disorder, symptoms of the disorder, or predisposition to the disorder. Treatment may also delay the onset, for example, prevent the onset, or prevent the worsening of the disease or condition.

[0144] Methods of administering kallikrein-binding antibodies and other agents are also described in "Pharmaceutical Compositions." The appropriate dose of the molecule used may depend on the age and weight of the subject, as well as the particular agent used. For example, antibodies may be used as competitive agents to inhibit or reduce undesirable interactions between plasma kallikrein and its substrates (e.g., factor XII or HMWK). The dose of the antibody may be sufficient to inhibit 90%, 95%, 99%, or 99% of the activity of plasma kallikrein in the patient, particularly at the disease site. This may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg / Kg. For an IgG (two binding sites) with a molecular weight of 150,000 g / mol, these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM binding sites for a blood volume of 5 L.

[0145] In one embodiment, the antibodies are used, for example, to inhibit the activity of plasma kallikrein in vivo (e.g., to inhibit at least one of the plasma kallikreins, e.g., to inhibit the production of factor XIIa and / or bradykinin). The binding proteins can be used by themselves or can be conjugated to agents such as cytotoxic drugs, cytotoxic enzymes, or radioisotopes.

[0146] Antibodies can be used directly in vivo to eliminate antigen-expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). The antibodies described herein may contain a complement-binding effector domain, such as the Fc portion from IgG1, IgG2, or IgG3 or the corresponding portion of IgM that binds complement. In one embodiment, a population of target cells is treated ex vivo with the antibodies described herein and appropriate effector cells. The treatment is supplemented by adding complement or serum containing complement. Furthermore, phagocytosis of target cells coated with the antibodies described herein can be improved by binding of complement proteins. In another embodiment, target cells coated with antibodies containing complement-binding effector domains are lysed by complement.

[0147] Methods for administering plasma kallikrein-binding antibodies are described in "Pharmaceutical Compositions." The appropriate dose of the molecule used depends on the age and weight of the subject and the particular drug used. The antibodies can be used, for example, as competitive drugs to inhibit or reduce undesirable interactions between natural or pathological drugs and plasma kallikrein.

[0148] A therapeutically effective amount of an antibody described herein can be administered to a subject having, suspected of having, or at risk for HAE to treat the disorder (e.g., alleviate or ameliorate symptoms or characteristics of the disorder, slow, stabilize, and / or halt progression of the disease).

[0149] The antibodies described herein can be administered in a therapeutically effective amount, which is an amount effective upon administration to a subject in one or more doses in treating the subject, e.g., curing, alleviating, ameliorating, or improving at least one symptom of a disorder in the subject to a degree greater than would be expected in the absence of such treatment.

[0150] Dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly preferred to formulate parenteral compositions in unit dosage form. Unit dosage form as used herein refers to physically discrete units suitable as unit dosage forms for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0151] In some embodiments, the antibody (e.g., DX-2930) is administered as a single dose, e.g., 0.1-3 mg / kg. In other embodiments, multiple doses, such as once every 1-4 weeks, e.g., every two weeks or monthly (e.g., every 28 days), are administered. The multiple doses may each range from 0.1-3 mg / kg. In some examples, the patient is administered multiple doses, e.g., once every 1-4 weeks, e.g., every two weeks or monthly, for an appropriate period of time, followed by maintenance therapy at the same or lower doses monthly or once every two months.

[0152] In some embodiments, the patient can be monitored before, after, or during the course of treatment for side effects (e.g., elevated levels of creatine phosphatase) and / or the level of inhibition of pKal by the antibody (e.g., serum or plasma concentration of the antibody, or pKal activity levels). If adverse effects are observed, the dose of the antibody may be reduced or treatment may be terminated. If the level of inhibition is below the minimum therapeutic level, an additional dose of the antibody may be administered to the patient.

[0153] In some embodiments, the plasma or serum concentration of the antibody (e.g., DX-2930) may be measured during the course of treatment (e.g., after an initial dose) to assess the efficacy of the treatment. If the plasma or serum concentration of the antibody is less than 80 nM, a follow-up dose is required, which may be the same as or higher than the initial dose. 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 obtained from the subject, for example, by immunoassay or MS assay. The plasma or serum concentration of the antibody may also be measured by determining the inhibition level of pKal in a plasma or serum sample obtained from a subject treated with the antibody. Such assays may include synthetic substrate assays or Western blot assays to measure cleaved kininogen as described herein.

[0154] Alternatively or additionally, plasma or serum levels of creatine kinase can be monitored during the course of treatment, and if plasma or serum levels of creatine kinase are found to increase during treatment, the dose of the antibody may be reduced or treatment may be terminated.

[0155] In some embodiments, the optimal dose (e.g., optimal prophylactic dose or optimal therapeutic dose) of an anti-pKal antibody (e.g., DX-2930 or an antigen-binding fragment thereof) described herein can be determined as follows: An initial dose of the antibody is administered to a subject in need of treatment. The plasma concentration of the antibody in the subject is measured. If the plasma concentration is less than 80 nM, the dose of the antibody is increased in subsequent administrations. The dose of the antibody that maintains a plasma concentration of the antibody greater than about 80 nM can be selected as the optimal dose for the subject. The subject's creatine phosphokinase levels can be monitored during the course of treatment, and the optimal dose for the subject can be adjusted based on creatine phosphokinase, for example, if an increase in creatine phosphokinase is observed during treatment, the dose of the antibody can be reduced.

[0156] Combination therapy The anti-plasma kallikrein antibodies described herein can be administered in combination with one or more other therapies to treat a disease or condition associated with plasma kallikrein activity, such as, for example, a disease or condition described herein. For example, the plasma kallikrein binding antibodies can be used therapeutically or prophylactically with surgery, another anti-plasma kallikrein Fab or IgG (e.g., another Fab or IgG described herein), another plasma kallikrein inhibitor, a peptide inhibitor, or a small molecule inhibitor. Examples of plasma kallikrein inhibitors that can be used in combination therapy with the plasma kallikrein binding antibodies described herein include, for example, the plasma kallikrein inhibitors described in International Patent Publication No. WO 95 / 21601 or WO 2003 / 103475.

[0157] One or more plasma kallikrein inhibitors can be used in combination with one or more plasma kallikrein binding antibodies described herein, for example, the combination may require a lower dose of the inhibitor, thereby reducing side effects.

[0158] The plasma kallikrein binding antibodies described herein can be administered in combination with one or more current therapies for treating HAE. For example, DX-2930 or a functional variant thereof described herein can be co-administered with a second anti-HAE therapeutic agent, such as ecallantide, a C1 esterase inhibitor (e.g., CINRYZE™), aprotinin (TRASYLOL®), and / or a bradykinin B2 receptor inhibitor (e.g., icatibant (FIRAZYR®)).

[0159] The term "combination" refers to the use of two or more agents or therapies to treat the same patient, where the use or action of the agents or therapies overlap in time. The agents or therapies can be administered simultaneously (e.g., as a single formulation administered to the patient or as two separate formulations administered simultaneously) or sequentially in any order. Sequential administration is administration administered at different times. The time between administration of one agent and administration of another agent can be minutes, hours, days, or weeks. The use of plasma kallikrein binding antibodies described herein can also be used to reduce the dose of another treatment, for example to reduce side effects associated with another agent being administered. Thus, combination can include administering the second agent at a dose at least 10, 20, 30, or 50% lower than would be used in the absence of the plasma kallikrein binding antibody.

[0160] The combination therapy may include administering an agent that reduces the side effects of another treatment, which may be an agent that reduces the side effects of a treatment for a plasma kallikrein-related disorder.

[0161] 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. Thus, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. Publications mentioned herein are incorporated by reference for any purpose or subject matter herein.

[0162] Working Example Example 1: Study of Single Ascending Doses of DX-2930 in Healthy Volunteers A single ascending dose in healthy volunteers was performed using the following doses of DX-2930: 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Data for each volunteer was obtained and analyzed to determine the safety of each dose. The complete and variable sequences of the heavy and light chains of DX-2930 are provided below, where the signal sequence is in italics. The CDRs are in bold and underlined. [ka] [ka] [ka] [ka] [Table 1]

[0163] Phase 1a Study Design The study was randomized, double-blind, and placebo-controlled. Single and ascending doses of DX-2930 were administered subcutaneously to healthy subjects. Participants were randomly assigned to cohorts of 1 to 4 subjects, each corresponding to a single dose (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg). Each cohort included six active-treated subjects and two placebo-treated subjects. After completing the dosing schedule, all subjects were monitored for 16 weeks.

[0164] Safety Results DX-2930 was well tolerated at single doses up to 3.0 mg / kg without any dose-limiting toxicities; thus, this study did not gather evidence of any clinically significant safety signals associated with DX-2930.

[0165] The clinical study results did not show any clinical imbalance between DX-2930 and placebo with respect to any adverse events. The most commonly reported adverse events included headache (25% of subjects treated with DX-2930 and 25% of subjects treated with placebo). There were no serious adverse events, and all adverse events resolved.

[0166] Analysis of vital signs, physical exam, and electrocardiograms (ECGs) indicated an upper respiratory tract infection in one subject receiving the 0.1 mg / kg dose. However, the investigators reported that this infection was mild and not related to treatment. No other abnormalities were observed in the vital signs, physical exam, or electrocardiograms (ECGs) of study subjects.

[0167] Testing for anti-drug antibodies did not yield evidence of seroconversion.

[0168] Only two subjects with serious AEs were reported as treatment-related by a blinded investigator. An elevation of creatine phosphokinase of 902 U / L (reference range: 21-215 U / L) was observed in a subject receiving 0.1 mg / kg DX-2930 (4.2% of all DX-2930-treated subjects). An elevation of creatine phosphokinase of 1967 U / L (reference range: 32-294 U / L) was observed in a subject receiving placebo (12.5% ​​of placebo-treated subjects). Results did not indicate lab abnormalities associated with any other AEs or findings that could suggest clinical significance. No subjects had injection site reactions. [Table 2] *Treatment-related AEs: AEs were assessed for their relevance to study drug by a blinded investigator. Note: Here, the term "adverse event" (AE) specifically refers to a treatment-emergent adverse event. An adverse event was considered to be treatment-emergent if its onset time was from after administration of study drug to the final post-dose follow-up visit at 112 days, or, for events whose onset time preceded study drug administration, if the AE increased in severity during the 112-day post-dose follow-up period.

[0169] Pharmacodynamic (PD) and pharmacokinetic (PK) outcomes Table 3 provides an assessment of the pharmacokinetic parameters for each dose cohort. Mean C max and AUC last The values ​​exhibit a precise linear dose-dependence consistent with a well-characterized antibody. A drug with a long half-life allows for a less frequent dosing schedule to achieve stable, steady-state blood levels. DX-2930 showed a consistent half-life extension of nearly 3 weeks across all dose groups.

[0170] Therapeutic candidates for the prevention of HAE should have long half-lives and predictable pharmacokinetic profiles to allow for infrequent dosing and logical technical dosing principles. DX-2930 offers a consistent pharmacokinetic profile, allowing the determination of dosing regimens that provide significant therapeutic benefit to HAE patients. [Table 3]

[0171] Pharmacokinetic (PK) parameters of DX-2930 were evaluated after a single dose in healthy subjects. Plasma drug concentrations above the target level of 80 nM were obtained after a single dose of 3 mg / kg. Drug levels around or above 80 nM were maintained for approximately 10 days (Figure 7). Drug levels continued to accumulate with repeated drug administration until a steady state was reached. Targeted drug levels above 80 nM were obtained and maintained for extended periods, even after only a single dose of DX-2930. The PK data from this study support the feasibility of a dosing strategy to obtain targeted plasma drug concentrations above 80 nM and then maintain those concentrations continuously. Furthermore, drug levels above 80 nM may be achieved as needed to obtain sufficient plasma kallikrein inhibition associated with the prevention of HAE.

[0172] A pharmacodynamic (PD) evaluation of DX-2930 was performed. To further characterize DX-2930, exploratory biomarker assays were performed ex vivo on subject plasma samples to evaluate the pharmacodynamic profile of the molecule. Two independent assays were performed - a plasma kallikrein activity assay using an artificial fluorogenic substance that measures the cleavage of kininogen, the natural substrate of plasma kallikrein from which bradykinin is generated, and a Western blot assay.

[0173] These assays are semi-quantitative. Thus, data points should be interpreted relative to other data points within an experiment, and not compared across assays or other assay systems. These assays are performed in plasma from normal subjects with normal levels of C1 inhibitor. As a result, the healthy subjects used in these biomarker assays do not develop attacks of HAE. The purpose of the evaluation of these biomarkers was to confirm that DX-2930 in plasma from treated subjects has inhibitory activity against plasma kallikrein. Equally important, these studies were performed to evaluate whether the pharmacodynamic results supported the observed PK profile.

[0174] FIG. 9 depicts the pharmacodynamic effects of DX-2930. Plasma from subjects treated with the test agent was activated ex vivo to induce the contact phase pathway, thereby stimulating plasma kallikrein production and activity. pKal activity was measured via a fluorogenic assay. Data from the 1 and 3 mg / kg groups are depicted. Inhibition of plasma kallikrein was particularly evident in the 1 and 3 mg / kg dose groups. No discernible inhibition was observed in the 0.1 mg / kg or placebo groups. The observed inhibition was both dose and time dependent and confirms the inhibitory activity of DX-2930. The PD effects of DX-2930 support the PK data of DX-2930.

[0175] Therapeutic Uses of DX-2930 For effective prevention, a prerequisite for efficacy is that the relevant drug target is continuously inhibited at a level above the minimum amount required. Gaps in the inhibition range should be minimized or avoided altogether. If the inhibition level falls below the minimum level required, the individual may be biologically unstable to the activation of the pathological process and may be at clinical risk for a disease event.

[0176] There are no widely established principles of prevention in HAE. In HAE, plasma kallikrein represents a validated drug target that is important in the pathogenesis of edema attacks. Preventing attacks of HAE may require continuous maintenance of inhibition of plasma kallikrein above the minimum therapeutic level. Gaps in coverage over time should be minimal to avoid vulnerable periods. This need is further emphasized by the phenomenon of positive feedback loops that have been hypothesized to play an important role in attacks of HAE. Once this cascade is initiated, activation of plasma kallikrein leads to activation of factor XII, which in turn drives the production of more plasma kallikrein.

[0177] The inhibition of plasma kallikrein by DX-2930 and ecallantide, a known therapeutic agent approved in the United States for the acute treatment of HAE attacks, was compared in an in vitro assay in which human plasma was exposed to an agent that initiates the contact phase and converts prekallikrein to active plasma kallikrein.

[0178] The peak concentration of ecallantide in plasma after administration to patients is 80 nM, providing only partial inhibition of plasma kallikrein activity. Around this concentration range of 80 nM, the results of these assays indicate that DX-2930 is as potent as ecallantide (Figure 5). If 80 nM appears to be the plasma kallikrein level suitable for HAE attacks, and if DX-2930 is as potent as ecallantide in inhibiting plasma kallikrein in this concentration range, then it can be hypothesized that maintaining DX-2930 above a plasma drug concentration of 80 nM on a sustained basis will prevent HAE attacks (Figure 6). A target plasma drug level of 80 nM for DX-2930 seems solid based on currently available data and understanding of disease biology. Although 80 nM is an initial target, lower or higher drug levels of DX-2930 may be necessary to obtain a therapeutic dose.

[0179] Example 2 Multiple Ascending Dose Study of DX-2930 in Patients with HAE Multiple doses of DX-2930 are administered to HAE patients at the following doses: 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Figure 2 provides the plasma concentrations predicted to be achieved after repeated administration of a 3 mg / kg dose of DX-2930. The initial concentration profile is consistent with that observed after single dose administration in healthy subjects.

[0180] PK modeling can be performed to predict the pharmacodynamic and pharmacokinetic properties of DX-2930 after chronic administration. Figure 8 depicts a hypothetical scenario in which DX-2930 is administered at 3 mg / kg every 28 days in healthy subjects. The results of this modeling suggest that after steady state is achieved, repeated administration will continually maintain drug concentrations around or above the initially targeted 80 nM level (see Example 2 for a discussion of this threshold).

[0181] The safety and efficacy of DX-2930 in HAE patients will be evaluated after two doses of DX-2930, one week apart. Biomarker data (along with pharmacokinetics) will be evaluated to determine which dose to use in a follow-up study to evaluate the efficacy of DX-2930 (at different doses) in preventing HAE attacks.

[0182] Example 3 Pharmacological Modeling of Dosing Regimens to Investigate Long-Term Prevention of Hereditary Angioedema method In vitro inhibition of pKal by ecallantide and DX-2930 was assessed using a synthetic substrate assay. Modeling was performed using pharmacokinetic (PK) data from a single ascending dose subcutaneous administration study of DX-2930 in healthy subjects.

[0183] result In vitro pKal assays showed that DX-2930 and ecallantide exhibited comparable pharmacodynamic (PD) activity at a concentration of 80 nM. Plasma concentrations of pKal inhibitors below 80 nM exhibited only partial preventive effects, providing further evidence for the medium- and high-dose hypotheses over the low-dose hypothesis. Thus, continuously maintaining DX-2930 drug levels above approximately 80 nM should achieve the inhibition level of pKal described by the medium-dose hypothesis. PK modeling indicates that chronic DX-2930 administration results in steady-state plasma drug concentrations of >80 nM with 100 mg every 2 weeks (or 300 mg every 4 weeks) and >200 nM with 300 mg every 2 weeks.

[0184] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic form of equivalent or similar feature.

[0185] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to suit various applications and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the claims.

Claims

[Claim 1] 1. A method of treating hereditary angioedema (HAE), comprising: administering to a subject in need thereof an antibody that binds active plasma kallikrein in an effective amount such that the plasma concentration of said antibody in said subject is greater than about 80 nM. A method comprising:

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