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

Abstract

Methods for treating hereditary angioedema are provided. The present invention provides plasma kallikrein binding proteins, such as antibodies that bind to active plasma kallikrein, and methods of using such proteins in the treatment of hereditary angioedema.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority on the filing dates of U.S. Provisional Patent Application No. 61 / 929,716 filed on 21 January 2014, U.S. Provisional Patent Application No. 61 / 944,361 filed on 25 February 2014, and U.S. Provisional Patent Application No. 62 / 021,397 filed on 7 July 2014. The entire contents of these referenced applications are incorporated herein by reference. [Background technology]

[0002] Plasma kallikrein is a type of contact-phase serine protease that may be a drug target for various inflammatory, cardiovascular, infectious (sepsis), and neoplastic diseases (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). The contact phase is activated by either factor XII upon exposure to exogenous or negatively charged surfaces, or by prolylcarboxypeptidase on the surface of endothelial cells (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies endogenous coagulation via the feedback activity of factor XII and enhances inflammation through the production of the pro-inflammatory nonapeptide bradykinin. As a major kininogenase in circulation, plasma kallikrein plays a significant role in bradykinin production in vascular structures. A genetic deficiency in C1 inhibitor protein (C1-INH), the main natural inhibitor of plasma kallikrein, results in hereditary angioedema (HAE). HAE patients often suffer from painful edema attacks triggered by unknown factors (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). [Overview of the project] [Problems that the invention aims to solve]

[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, which maintains a plasma concentration of the antibody above 80 nM, is sufficient to exhibit a beneficial (e.g., prophylactic) effect in the treatment of hereditary angioedema. Furthermore, administering DX-2930 at a dose of 100 mg every two weeks, or at a dose of 300 mg every four weeks, maintains a steady-state plasma drug concentration above 80 nM, and administering DX-2930 at a dose 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 finding that antibodies that bind to the active form of human plasma kallikrein exhibited no dose-limiting toxicity findings at single doses up to 3.0 mg / kg, and showed excellent therapeutic effects 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). Pharmacokinetic results showed that DX-2930 had linear dose-dependent exposure across dose groups and a mean elimination half-life of 17 to 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] Accordingly, one aspect of the present disclosure features a method of treating HAE (e.g., Type I, Type II, or Type III), comprising administering to a subject in need thereof an antibody that binds the active form of pKal in an effective amount (e.g., 100 mg to 400 mg, or 100 to 300 mg) such that the 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 present disclosure is a method of treating HAE (e.g., Type I, Type II, or Type II), comprising (a) administering to a subject in need thereof an antibody that binds active plasma kallikrein (e.g., a full-length antibody or an antigen-binding fragment thereof) at a first dose, (b) measuring the plasma concentration of the antibody in the subject, and (c) administering the antibody to the subject at a second dose when the plasma concentration of the antibody is less than about 80 nM. In some embodiments, the first dose, the second dose, or both is 100 mg to 400 mg or 100 mg to 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] Accordingly, one aspect of the present disclosure features a method of treating HAE (e.g., Type I, Type II, or Type III), comprising administering a single dose of an isolated antibody to a subject in need thereof, wherein 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 comprises monitoring the level of creatine phosphokinase in the subject before and after the treatment. In some embodiments, the single dose of any of the antibodies described herein is 0.1 to 3 mg / kg (e.g., 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg).

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

[0009] In a further different embodiment, the Disclosure provides a method for treating HAE, comprising: (i) administering one or more doses of an isolated antibody (e.g., a full-length antibody or its antigen-binding fragment) conjugating active plasma kallikrein (e.g., an antibody that conjugates human active plasma kallikrein but not human prekallikrein) to a subject in need; (ii) measuring the level of inhibition of plasma kallikrein by the antibody in the subject after the last dose; and (iii) administering further doses of the antibody to the subject if the inhibition level is lower than a minimum therapeutic level. In some embodiments, one or more doses are 0.1 to 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 less than about 80 nM.

[0010] In any of the methods described herein, the anti-pallikrein antibody may be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, the antibody binds to active plasma kallikrein but not to 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 the same heavy chain CDR as DX-2930, the same light chain CDR as DX-2930, or both. In some embodiments, the antibody is DX-2930, which is the 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 subcutaneously. In some embodiments, the subjects are human patients who are suffering from HAE attacks, suspected of having HAE attacks, or at risk of HAE attacks. 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 includes monitoring the level of creatine phosphokinase in the subject before and after treatment, or during the course of treatment. If an increase in creatine phosphokinase is observed, the dose of the antibody (e.g., DX-2930) may be reduced, or treatment may be terminated.

[0014] In a further different aspect, the Disclosure provides a method for determining an optimal dose (e.g., an optimal prophylactic dose) for treating hereditary angioedema (HAE) in a subject, comprising: (a) administering (e.g., subcutaneously) an antibody described herein (e.g., DX-2930 or its antigen-binding fragment) conjugating active plasma kallikrein to the subject in need in an initial dose; (b) measuring the 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 the dose that maintains the 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 of administration of the antibody. 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 method described above may further include monitoring the level of creatine phosphokinase in the subject before and after treatment, or during the course of treatment. Furthermore, the method may further include reducing the antibody dose or discontinuing treatment if an increase in creatine phosphokinase is observed.

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

[0017] Also within the scope of this disclosure are (a) a pharmaceutical composition for use in treating HAE or determining the optimal dose of a drug for treating HAE, comprising one of the anti-kallikrein antibodies described herein and a pharmaceutically acceptable carrier, and (b) the use of a pharmaceutical composition for manufacturing a drug for treating HAE. The use of the antibody for the intended purpose can be carried out under the administration conditions described herein.

[0018] Details of one or more embodiments of the present invention are described below. Other features or advantages of the present invention will become apparent from the following drawings, the detailed description of some embodiments, and the appended claims. [Brief explanation of the drawing]

[0019] [Figure 1] This graph shows the mean concentrations of DX-2930 after subcutaneous (SC) administration to healthy subjects. The concentration-time plots for each dose cohort are shown on a logarithmic scale. Error bars represent standard deviations. This profile indicates linear, dose-dependent exposure. The parallel efflux phases across dose groups are consistent with a normally functioning antibody, exhibiting dose-independent kinetics where all doses act uniformly. [Figure 2] This graph shows the predicted plasma concentrations after repeated administration of DX-2930 at a dose of 3 mg / kg via subcutaneous injection every 28 days in healthy subjects. [Figure 3] This graph illustrates the general principles of drug use for the prevention of disease conditions. [Figure 4] This graph illustrates the general principles of using DX-2930 for the prevention of HAEs. [Figure 5] This graph shows the comparative inhibitory activity of DX-2930 and ecalantide against pKal in vitro. [Figure 6] This graph represents alternative hypotheses regarding the conditions necessary to continuously maintain plasma concentrations of DX-2930 above 80 nM for the treatment and / or prevention of HAEs. Alternatively, the plasma concentrations of DX-2930 required for the treatment and / or prevention of HAEs may be lower or higher than 80 nM. [Figure 7] This graph shows that plasma drug levels of 80 nM or higher can be achieved after a single dose of 3 mg / kg of DX-2930. [Figure 8] This graph shows pharmacokinetic (PK) modeling of chronic DX-2930 administration, delivered subcutaneously at a dose of 3 mg / kg every 28 days, in healthy subjects. [Figure 9] This graph shows the pharmacodynamic (PD) effect of DX-2930 at a dose of 3 mg / kg SC every 28 days in healthy subjects. [Figure 10] This graph shows pharmacodynamic (PD) and pharmacokinetic (PK) data after a single dose (3 mg / kg) of DX-2930. 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-value < 0.05 (Day 5, 3 mg / kg vs. placebo). [Figure 11] This graph shows the pharmacodynamic (PD) activity of DX-2930 against natural biological substrates (HMWK cleavage was measured as the formation of two-chain HMWK). [Figure 12] This graph shows the sustained physiological activity of DX-2930 over time after a single dose of 3 mg / kg. *P-value < 0.05 (3 mg / kg vs. prophylactic dose on day 28) [Modes for carrying out the invention]

[0020] definition Before further describing the present invention, for convenience, specific terms used in this specification, examples, and appended claims are defined individually. Other terms are defined as will become clear in this specification.

[0021] The singular forms "a," "an," and "the" include the plural form unless otherwise specified.

[0022] The term “antibody” refers to a protein containing at least one immunoglobulin variable domain (variable region) or immunoglobulin variable domain (variable region) sequence. For example, an antibody may contain a heavy chain (H) variable region (abbreviated herein as VH or HV) and a light chain (L) variable region (abbreviated herein as VL or LV). In another example, an antibody may contain two heavy chain (H) variable regions and two light chain (L) 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 the structural features of IgA, IgG, IgE, IgD, IgM (and their subtypes). The antibodies may be derived from any source, but antibodies from primates (human and non-human primates) and primate-like antibodies are preferred.

[0023] The VH and VL regions can be further divided into more conserved regions called "framework regions (FRs)" and highly variable regions called "complementarity-determining regions (CDRs)." The degree of framework regions and CDRs is 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). Kabat's definition is used herein. Each VH and VL generally consists of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.

[0024] As used herein, “immunoglobulin variable domain sequence” refers to an amino acid sequence capable of forming an immunoglobulin variable domain structure such that one or more CDR regions are positioned in a conformation suitable for an antigen-binding site. For example, the sequence may include all or part 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, include one or more inserted or additional terminal amino acids, or include other modifications. In one embodiment, a polypeptide containing an immunoglobulin variable domain sequence can bind to another immunoglobulin variable domain sequence to form a structure that preferentially interacts with an antigen-binding site, such as plasma kallikrein.

[0025] The VH or VL chains of an antibody can form heavy or light immunoglobulins, respectively, by further including all or part of the constant region of the heavy or light chain. In one embodiment, the antibody is a tetramer of two heavy immunoglobulins and two light immunoglobulins, where the heavy and light immunoglobulins are internally linked, for example, by disulfide bonds. In IgG, the heavy chain constant region includes the domains CH1, CH2, and CH3 of the three immunoglobulins. The light chain constant region includes the CL domain. The variable regions of the heavy and light chains include binding domains that interact with the 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 first complement (Clq) of the classical complement pathway. The light chain of the immunoglobulin may be kappa or lambda. In one embodiment, the antibody is glycosylated. Antibodies may be functional with respect to antibody-dependent cytotoxicity and / or complement-mediated cytotoxicity.

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

[0027] All or part of an antibody may be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include those for the constant regions of kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu, as well as genes for many immunoglobulin variable regions. A full-length immunoglobulin "light chain" (approximately 25 kDa or approximately 214 amino acids) is encoded by a gene for the variable region at the NH2 terminus (approximately 110 amino acids) and a gene for the constant region of kappa or lambda at the COOH terminus. Similarly, a full-length immunoglobulin "heavy chain" (approximately 50 kDa or approximately 446 amino acids) is encoded by a gene for the variable region (approximately 116 amino acids) and one of the other constant region genes mentioned above, such as gamma (encoding approximately 330 amino acids). Since HC CDR3 varies from approximately 3 amino acid residues to more than 35 amino acid residues, the length of human HC varies considerably.

[0028] The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that possess 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 fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments bound by disulfide crosslinking at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the VL and VH domains of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that can be constructed as a single protein chain, where the pair of VL and VH regions form a monovalent molecule known as single-chain Fv (scFv). See, for example, U.S. Patents 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 the prior art known to those skilled in the art. The term “monospecific antibody” refers to an antibody that exhibits a single binding specificity and affinity to a specific target, such as an epitope. The term includes “monoclonal antibody” or “monoclonal antibody composition” and refers to a preparation of an antibody or its fragment in a single molecular composition, regardless of how the antibody was produced, as used herein.

[0030] An antibody is "germlined" by reverting one or more non-germline amino acids in the framework regions to the corresponding germline amino acids of the antibody, so long as the binding properties are substantially retained.

[0031] The inhibition constant (Ki) provides a measure of inhibitor potency, which is the concentration of inhibitor required to reduce enzyme activity by half and is not dependent on enzyme or substrate concentrations. The apparent Ki (K i,app ) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of the inhibitor (e.g., an inhibitory binding protein) on the extent of the reaction (e.g., enzyme activity); fitting the change in pseudo-first order rate constant as a function of inhibitor concentration to the output of Morrison's equation (Equation 1) provides an estimate of the apparent Ki value. The Ki is obtained from the y-intercept extracted from a linear regression analysis of a plot of K i,app versus substrate concentration. [Formula] Equation 1 wherein, v = measured rate; v0 = rate in the absence of inhibitor; K i,app = apparent inhibition constant; I = total inhibitor concentration; and E = total enzyme concentration.

[0032] As used herein, "binding affinity" refers to the apparent binding constant or K A . K A is the reciprocal of the dissociation constant (K D ). A binding antibody may, for example, have a binding affinity of at least 10 for a particular target molecule, such as plasma kallikrein 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 and 10 11 M -1 . Higher affinity binding of a binding antibody to a first target compared to a second target is indicated by a higher K for binding to the first target than the K for binding to the second target A (or the numerical K D ) for binding to the second target, where the K for binding to the first target is higher A(or a smaller number K) D This can be expressed by ). In such cases, the binding antibody has specificity for the first target (e.g., the protein of the first three-dimensional structure or its mimicry) compared to the second target (e.g., the same protein of the second three-dimensional structure or its mimicry). The difference in binding affinity (e.g., with respect to specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 It can 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 fluorescence assays). 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 concentrations of bound and free binding proteins as a function of the concentration of the binding protein (or target). The following equation shows that the concentration of bound binding protein ("Bound") is related to the concentration of free binding protein ("Free") and the concentration of the binding site with respect to the binding protein on the target:

number

[0034] However, the exact K A Making such a decision is not always necessary, and in some cases, K AThis is because it is sufficient to obtain quantitative measurements of affinity, which are proportional to the activity of the substance, and can be used for comparison to determine, for example, whether a higher affinity is, for example, twice as high, or to obtain quantitative measurements of affinity or inferences of affinity by functional assays such as in vitro or in vivo assays.

[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 in particular 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 this antibody and under identical conditions.

[0036] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined by those skilled in the art. These families include amino acids having 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), β-partitioned 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 contain one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) compared to the binding proteins described herein. Plasma kallikrein-binding proteins may have mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) (e.g., at least one, two, three, or four, and / or fewer than 15, fewer than 12, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, or fewer than 2) compared to the proteins described herein, such as mutations that have no substantial effect on the function of the protein. These mutations may be located in the framework region, the CDR, and / or the constant region. In some embodiments, the mutations are located in the framework region. In some embodiments, the mutations are located in the CDR. In some embodiments, the mutations are located in the constant region. Whether a particular substitution is acceptable, that is, whether it does not adversely affect biological properties such as binding activity, can be predicted, for example, by evaluating whether the mutation is conserved, or by the method described in Bowie, et al. (1990) Science 247:1306-1310.

[0038] A “virtually human” immunoglobulin variable region is an immunoglobulin variable region that contains a sufficient number of human framework amino acid positions so as not to induce an immunogenic response in a normal human. A “virtually human” antibody is an antibody that contains a sufficient number of human amino acid positions so as not to induce an immunogenic response in a normal human.

[0039] An "epitope" refers to a site on a target compound to which a binding protein (such as an antibody, like a Fab or full-length antibody) is bound. If the target compound is a protein, this site can consist entirely of amino acid components, entirely of chemically modified amino acids of the protein (such as glycosyl moieties), 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] The first binding antibody "binds to the same epitope" as the second binding antibody if it binds to the same site on the target compound to which the second binding antibody binds, or to a site that overlaps with the site to which the second binding antibody binds (for example, overlapping by 50%, 60%, 70%, 80%, 90%, or 100% in terms of amino acid sequence or other molecular features (e.g., glycosyl groups, phosphate groups, or sulfate groups)).

[0041] If the binding affinity of the first binding antibody to the epitope of the first binding antibody reduces the amount of the second binding antibody that binds to the epitope of the second binding antibody (for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more), then the first binding antibody "competes with the second binding antibody for binding." This competition can be direct (for example, the first binding antibody binds to an epitope identical to or overlapping with the epitope bound by the second binding antibody) or indirect (for example, the binding affinity of the first binding antibody to the epitope of the first binding antibody causes a steric change in the target compound that reduces the properties of the second binding antibody that binds to the epitope of the second binding antibody).

[0042] The calculation of “homology” or “sequence identity” (these terms are used interchangeably herein) between two sequences is performed as follows: These sequences are aligned to optimize the comparison (for example, 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, with a Blosum62 score matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Next, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, “identity” of an amino acid or nucleic acid is equivalent to “homology” of an amino acid or nucleic acid). The percentage identity between the two sequences is a function of the number of identical positions shared in these 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 include a sufficient number of amino acid positions from the human framework so that it does not induce an immunogenic response in normal humans. Examples of “humanized” immunoglobulins include U.S. Patent No. 6,407,213 and U.S. Patent No. 5,693,762.

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

[0046] The “patient,” “subject,” or “host” (these terms are used interchangeably) treated by the methods described herein may mean either a human or a non-human animal.

[0047] The terms “prekallikrein” and “preplasma kallikrein” are used interchangeably herein and refer to the enzyme precursor form of active plasma kallikrein, also known as prekallikrein.

[0048] As used herein, “substantially identical” (or “substantially homologous”) means a first amino acid or nucleic acid sequence that contains a sufficient number of amino acid residues or nucleotides identical or equivalent to the second amino acid or nucleic acid sequence (e.g., having similar side chains, e.g., having conserved amino acid substitutions) such that the first and second amino acid or nucleic acid sequences have similar activity, e.g., binding activity, binding priority, or biological activity (or encode proteins having similar activity). In the case of an antibody, the second antibody has the same specificity as the same antigen and has at least 50%, at least 25%, or at least 10% of the affinity.

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

[0050] Furthermore, substantial identity exists with respect to the complementary strand when nucleic acid segments hybridize under selective hybridization conditions (e.g., very strict hybridization conditions). Nucleic acids may exist in whole cells, in cell lysates, or in partially or substantially purified forms.

[0051] Statistical significance can be determined by any method known to those skilled in the art. Exemplary statistical tests include the Student's t-test, the non-parametric Mann-Whitney U test, and the non-parametric Wilcoxon test. Some statistically significant relationships have p-values ​​less than 0.05 or 0.02. Certain binding proteins may exhibit differences in specificity or binding ability that are statistically significant (e.g., p-value < 0.05 or 0.02). For example, terms such as “inducing,” “inhibiting,” “enhancing,” “increasing,” “increasing,” and “decreasing” may refer to differences, such as statistically significant differences between two states, indicating a distinguishable qualitative or quantitative difference between them.

[0052] A "therapeutably 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 an untreated control. The properties of a compound that modulates a measurable parameter, such as a disease-related parameter, can be evaluated in animal model systems that predict the effects in human disorders and pathologies. Alternatively, this property of a composition can be evaluated by testing the properties of the parameter-modulating compound in vitro.

[0053] As used herein, the term “to treat” means the application or administration of a composition comprising one or more active agents to a subject having an allergic disease, symptoms of an allergic disease, or a predisposition to an allergic disease, for the purpose of curing, alleviating, mitigating, altering, remedying, improving, or influencing a disease, symptoms of a disease, or predisposition to a disease. “Prophylate treatment,” also known as “preventive treatment,” refers to treatment aimed at protecting a patient from a disease to which they are or may be exposed, or reducing the risk of such disease.

[0054] The term "preventing" a disease in a subject refers to administering a drug that prevents at least one of the diseases, i.e., administering it before the clinical symptoms of an undesirable condition (e.g., a disease or other undesirable state in the host animal) appear, so that the host does not develop the undesirable condition. "Preventing" a disease may also refer to "preventive drugs" or "preventive treatments."

[0055] A "prophylactically effective dose" refers to the amount that is effective in the required dosage and for the duration necessary to achieve the desired prophylactic outcome. Generally, a prophylactically effective dose is less than a therapeutically effective dose, because prophylactic doses are used for subjects before or during the early stages of the disease.

[0056] Plasma kallikrein-conjugated antibodies The plasma kallikrein-conjugated antibody 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 consist only of antigen-binding fragments (e.g., Fab, F(ab')2, or scFv fragments). The conjugated antibody may consist of two heavy-chain immunoglobulins and two light-chain immunoglobulins, or it may be a single-chain antibody. The plasma kallikrein-conjugated antibody may be a recombinant protein such as a humanized antibody, a CDR graft antibody, a chimeric antibody, a deimmunized antibody, or an antibody produced in vitro, and may optionally contain a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein-conjugated antibody is a monoclonal antibody.

[0057] In one embodiment, 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, such as human plasma kallikrein and / or mouse kallikrein.

[0058] The antibody may include one or more of the following features: (a) a human CDR or human framework region; (b) an HC immunoglobulin variable domain sequence comprising at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or one or more (e.g., one, two, or three) CDRs that are 100% identical to the HC variable domain CDR described herein; (c) an LC immunoglobulin variable domain sequence comprising at least 85, 88, (d) The LC immunoglobulin variable domain sequence comprises one or more (e.g., one, two, or three) CDRs that are identical to 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the LC variable domains described herein (e.g., in the whole or in the framework region or CDR); (e) The HC immunoglobulin variable domain sequence comprises one or more (e.g., one, two, or three) CDRs that are identical to 89, 90, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the LC variable domains described herein. (f) The C variable domain is identical to at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (for example, in the whole or in the framework region or CDR); (g) The antibody binds to the epitope conjugated by the antibody described herein, or competes for binding with the antibody described herein; (h) The primate CDR or primate framework region; (g) The HC immunoglobulin variable domain sequence is identical to the CDR1 of the HC variable domain described herein, and is less than 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (for example, in the whole or in the framework region or CDR); (g) The antibody binds to the epitope conjugated by the antibody described herein, or competes for binding with the antibody described herein; (h) The primate CDR or primate framework region; (g) The HC immunoglobulin variable domain sequence is identical to the CDR1 of the HC variable domain described herein, and less than 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (for example, in the whole or in the framework region or CDR); (g) The antibody binds to the epitope conjugated by the antibody described herein, or competes for binding with the antibody described herein; (g) The primate CDR or primate framework region; (h) The HC immunoglobulin variable domain sequence is identical to the CDR1 of the HC variable domain described herein, and less than 85, 88, 89, 90, 91, 92, 93, 94, 9 (i) The HC immunoglobulin variable domain sequence includes a CDR1 that differs by at least one amino acid and by two or three or fewer amino acids; (j) The HC immunoglobulin variable domain sequence includes a CDR2 of the HC variable domain described herein and a CDR2 that differs by at least one amino acid and by two, three, four, five, six, seven, or eight or fewer amino acids; (j) The HC immunoglobulin variable domain sequence includes a CDR3 of the HC variable domain described herein and a CDR3 that differs by at least one amino acid and by two, three, four, five, or six or fewer amino acids;(k) The LC immunoglobulin variable domain sequence comprises CDR1 of the LC variable domain described herein and CDR1 which differs by at least one amino acid and by 2, 3, 4, or 5 or fewer amino acids; (l) The LC immunoglobulin variable sequence comprises CDR2 of the LC variable domain described herein and CDR2 which differs by at least one amino acid and by 2, 3, or 4 or fewer amino acids; (m) The LC immunoglobulin variable domain sequence comprises CDR3 of the LC variable domain described herein and CDR1 which differs by at least one amino acid and by 2, 3, 4, or 5 or fewer amino acids (n) The LC immunoglobulin variable domain sequence differs from the LC variable domain described herein by at least one amino acid, and by 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids (e.g., in the whole, the framework region, or the CDR); and (o) the HC immunoglobulin variable domain sequence differs from the HC variable domain described herein by at least one amino acid, and by 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids (e.g., in the whole, the framework region, or the CDR).

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

[0060] Plasma kallikrein-binding antibodies may further inhibit plasma kallikrein, such as human plasma kallikrein.

[0061] In some embodiments, the plasma kallikrein-conjugating antibody does not bind to 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 to the active site or near the active site of the catalytic domain or fragment thereof of plasma kallikrein, or to an epitope that overlaps with the active site of plasma kallikrein.

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

[0064] The antibody is at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , and 10 11 M -1 With its binding affinity, it can bind to plasma kallikrein, for example, human plasma kallikrein. In one embodiment, the antibody is 1 × 10 -3 , 5×10 -4 s -1 , or 1 × 10 -4 s -1 Slower K off It binds to human plasma kallikrein. In one embodiment, the antibody is 1 × 10⁶ 2 , 1 x 10 3 , or 5×10 3 M -1 s -1 Faster K onThe antibody then binds to human plasma kallikrein. In one embodiment, the antibody binds to plasma kallikrein, but not to tissue kallikrein and / or plasma prekallikrein as much as it binds to plasma kallikrein (for example, the antibody binds to tissue kallikrein and / or plasma prekallikrein with a weak effect (e.g., 5, 10, 50, 100, or 100 times less than a negative control, or no effect at all)).

[0065] In one embodiment, the antibody is, for example, 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , and 10 -10 Antibodies inhibit human plasma kallikrein activity with Ki values ​​less than M. These antibodies exhibit IC50 values ​​less than, for example, 100 nM, 10 nM, 1, 0.5, or 0.2 nM. 50 Antibodies may have the following properties: For example, an antibody may modulate the activity of plasma kallikrein and the production of factor XIIa (e.g., derived from factor XII) and / or bradykinin (e.g., derived from high molecular weight kininogen (HMWK)). An antibody may inhibit the activity of plasma kallikrein and / or the production of factor XIIa (e.g., derived from factor XII) and / or bradykinin (e.g., derived from high molecular weight kininogen (HMWK)). The affinity of an antibody for human plasma kallikrein is less than 100 nM, less than 10 nM, less than 5 nM, less than 1 nM, and less than 0.5 nM of K. D This can be characterized as follows: In one embodiment, the antibody inhibits plasma kallikrein, but does not inhibit tissue kallikrein to the same extent as it inhibits plasma kallikrein (for example, the antibody inhibits tissue kallikrein with a weak effect (for example, 5, 10, 50, 100, or 1000 times less than a negative control, or no effect at all)).

[0066] In some embodiments, the antibody has an apparent inhibition constant (K) of 1000, 500, 100, 5, 1, 0.5, or less than 0.2 nM. i,app ) has.

[0067] Plasma kallikrein-conjugated antibodies may have their HC and LC variable domain sequences contained in a single polypeptide (e.g., scFv), or they may have their HC and LC variable domain sequences on different polypeptides (e.g., 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 IgG1, IgG2, IgG3, or IgG4. The antibody may be a soluble Fab. In other implementations, the antibody comprises Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecules containing one antigen-binding site of the binding protein as specified 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 antibody or a humanized antibody, or is non-immunogenic in humans. For example, the antibody contains one or more human antibody framework regions, e.g., all human framework regions, or framework regions that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to human framework regions. In one embodiment, the antibody contains a human Fc domain, or an Fc domain that is 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 a primate-like antibody, or is non-immunogenic in humans. For example, the antibody contains 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, or 99% identical to primate framework regions. In one embodiment, the antibody contains 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 (bonobos)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0071] In some embodiments, the affinity of primate antibodies to 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, and less than 0.5 nM of K D For example, characterized by less than 10 nM, less than 1 nM, or less than 0.5 nM.

[0072] In certain embodiments, the antibody does not contain sequences derived from mice or rabbits (for example, it 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 an antibody that conjugates the same epitope as DX2930, or competes with DX-2930 for binding to active plasma kallikrein.

[0074] In one example, a functional mutant of DX-2930 contains the same complementarity-determining region (CDR) as DX-2930. In another example, a functional mutant of DX-2930 contains the same V H and VL Compared to FR, V H or V L One or more mutations (e.g., conservative substitutions) may be present in any of the FRs. Preferably, such mutations do not occur in residues that are expected 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 another embodiment, the functional variant of DX-2930 is V H V contains at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical amino acid sequences. H Chain, and / or V of DX-2930 L V has at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) the same amino acid sequence. L The chain may also be included. These variants can bind to the active form of plasma kallikrein, and preferably do not bind to prekallikrein.

[0075] The "percent identicality" of two amino acid sequences is determined using the algorithm from 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 algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the target protein molecule. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used.

[0076] Antibody preparation The antibodies capable of conjugating PKal described herein can be prepared by any method known to those skilled in the art. For example, see 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 techniques. A full-length target antigen or fragment thereof, optionally bound to a carrier protein such as KLH, can be used to immunize a host animal to produce antibodies that bind to that antigen. The pathways and schedules for immunizing the host animal generally build upon established prior art for antibody stimulation and production, as further described herein. General techniques for the production of mouse antibodies, humanized antibodies, and human antibodies are known to those skilled in the art and are described herein. Antibodies of any mammalian subject, including humans, or antibodies that produce cells from them, can be manipulated to act as a basis for the production of mammalian cell lines, including human hybridoma cell lines. Generally, a host animal is seeded with a certain amount of immunogen, including those described herein, intraperitoneally, intramuscularly, orally, subcutaneously, plantarly, and / or intradermally.

[0078] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique described in 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 strains, including, but not limited to, X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used for hybridization. Generally, this 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, with or without serum supplementation, can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion techniques, EBV immortalized B cells may be used to produce the anti-PKal monoclonal antibodies described herein. If necessary, hybridomas may be expanded and subcloned, and the supernatant may be assayed for anti-immunogen activity by conventional immunoassay methods (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

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

[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 an expression or growth vector. The sequence encoding the antibody of interest may be maintained in a vector in a host cell, and the host cell may then be expanded and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic manipulation to “humanize” the antibody or to improve the antibody’s affinity (affinity maturation) or other characteristics. For example, the constant region may be manipulated to more closely resemble the human constant region in order to avoid an immune response when the antibody is used in human clinical trials and therapy. Genetically manipulating the antibody sequence may be preferable to obtain higher affinity for the target antigen and higher efficacy in terms of inhibiting PKal activity. It is apparent to those skilled in the art that one or more polynucleotides in an antibody can be altered while still maintaining its binding specificity to the target antigen.

[0081] In other embodiments, complete human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic mice, designed to produce a more desirable (e.g., completely human antibodies) or more potent immune response, may also be used to produce humanized or human antibodies. Examples of such techniques include Xenomouse® from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse® from Medarex, Inc. (Princeton, NJ). In another alternative example, antibodies may be produced by recombinant DNA using phage display or yeast technology. See, for example, U.S. Patents 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, using phage display technology (McCafferty et al., (1990) Nature 348:552-553), human antibodies and antibody fragments can be produced in vitro from an immunoglobulin variable (V) domain gene repertoire derived from unimmunized donors.

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

[0083] Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. For instance, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, 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 monoclonal antibodies in recombinant host cells. See, for example, International Patent Publication No. 87 / 04462. Next, DNA can be modified, for example, by substituting the coding sequence with the constant domains of human heavy and light chains in place of homologous mouse sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This method allows for the preparation of genetically modified antibodies, such as "chimeric" or "hybrid" antibodies, that have binding specificity to a target antigen.

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

[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 variable regions of the VH and VL of a parental non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known to those skilled in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structure are identified using the same molecular modeling analysis. In parallel, human VH and VL chains with amino acid sequences homologous to the parental non-human antibody are identified from any antibody gene database using the parental VH and Vl sequences as search queries. Then, human VH and VL acceptor genes are selected.

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

[0087] Single-chain antibodies can be prepared via recombination techniques by linking nucleotide sequences encoding a heavy-chain variable region and a light-chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, a phage or yeast scFv library can be produced by adapting the techniques described for single-chain antibody production (U.S. Patents 4,946,778 and 4,704,692), and PKal-specific scFv clones can be identified from the library using routine methods. Positive clones can be further screened to identify those that inhibit PKal activity.

[0088] Antibodies obtained according to methods known to those skilled in the art and those described herein can be characterized using methods well known to those skilled in the art. One such method is to identify the epitopes to which the antibody binds, or "epitope mapping." There are many methods known to those skilled in the art for mapping and characterizing the location of epitopes on proteins, including analysis of the crystalline structure of antibody-antigen complexes, 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 sequences to which the antibody binds. Epitopes may be linear epitopes, i.e., they may be structural epitopes formed by three-dimensional interactions of amino acids, which may or may not necessarily be contained within a single compartment of amino acids (a linear sequence of the primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids) can be isolated or synthesized (e.g., by recombination) and used in antibody binding assays. In another example, the epitopes to which antibodies bind can be determined by systematic screening using duplicate peptides derived from the target antigen sequence and determining the binding affinity with the antibody. Gene fragment expression assays fragment an open reading frame encoding the target antigen either randomly or by specific genetic construction, and determine the reactivity of the expression fragments of the antibody and antigen being tested. The gene fragments may be produced by PCR, for example, in the presence of radioactive amino acids, and then transcribed in vitro and translated into proteins. The binding of the antibody to the radioactively labeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Alternatively, specific epitopes can be identified by using a large library of random peptide sequences presented on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can be tested for binding affinity to a test antibody in a simple binding assay. Further examples include antigen-binding domain mutagenesis, domain exchange experiments, and alanine scan mutagenesis to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain exchange experiments can be performed using variants of a target antigen in which various fragments of the PKal polypeptide are substituted (exchanged) with sequences from closely related but antigenically different proteins (such as another member of the neurotrophin protein family). The importance of a particular antigen fragment to antibody binding affinity can be assessed by evaluating the antibody's binding affinity to the mutant PKal (e.g., the variant described in Example 2 below).

[0089] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether one antibody binds to the same epitope as another antibody. Competitive assays are well known to those skilled in the art.

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

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

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

[0093] Generally, a nucleic acid sequence encoding a protein is cloned into a nucleic acid expression vector. Naturally, if a protein contains multiple polypeptide chains, each chain can be cloned into an expression vector, for example, the same or different vectors, and they can then be expressed in the same or different cells.

[0094] For example, several antibodies, such as Fab, 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 instance, if Fab is encoded by a sequence in a phage display vector that includes a repressible stop codon between the display entity and the bacteriophage protein (or fragment thereof), the nucleic acid of the vector can be introduced into bacterial cells that cannot repress the stop codon. In this case, Fab is not fused to the gene III protein and is secreted into the periplasm and / or culture medium.

[0095] Furthermore, antibodies can also be produced in eukaryotic cells. In one embodiment, the antibody (e.g., scFv's) is expressed in yeast cells such as Pichia (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.Those skilled in the art would know, for example, oxygen conditions (see, e.g., Baumann K., et al. 2010 BMC Syst. Biol. 4:141), molar osmotic pressure (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), and 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 factors such as Bowdish, K. et al. 1991 J Biol Chem 266(18):11901-8, protein overexpression to enhance antibody production (see, for example, Gasser, B. et al., 2006 Biotechol. Bioeng. 94(2):353-61), culture acidity level (see, for example, Kobayashi H., et al., 1997 FEMS Microbiol Lett 152(2):235-42), and concentration of substances and / or ions (see, for example, Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):41-8). Furthermore, yeast systems can be used to produce antibodies with extended half-lives (see, for example, 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 the clonal antibody or its antigen-binding fragment include Chinese hamster ovary (CHO cells) (containing dhfr-CHO, as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 and used with a DHFR-selectable marker as described, for example, Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(1-2):65-80), and cells derived from transgenic animals, such as transgenic mammals. For example, the cells are mammalian epithelial cells.

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

[0098] In addition to nucleic acid sequences encoding diversified immunoglobulin domains, recombinant expression vectors may also include additional sequences such as sequences that control vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, generally selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (dhfr in the selection / amplification of methotrexate). - Includes (for use in host cells) and neo genes (for G418 selection).

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

[0100] In antibodies containing an Fc domain, the antibody production system may produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of the IgG molecule is glycosylated at asparagine 297 in the CH2 domain. This asparagine is a modification site with a branched oligosaccharide. This glycosylation has been shown to be necessary for effector function mediated by the Fcg receptor 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 post-translational modifications from other eukaryotes.

[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 includes a milk-specific promoter, a nucleic acid encoding the antibody of interest, and a signal sequence for secretion. The milk produced by a female transgenic mammal contains the antibody of interest secreted therein. The antibody can be purified from the milk or used directly for several applications.

[0102] Pharmaceutical composition One or more antibodies described herein may be present in a composition, for example, a pharmaceutically acceptable composition or a pharmaceutical composition. Plasma kallikrein-conjugated antibodies can be formulated with a pharmaceutically acceptable carrier. In some embodiments, 100 mg to 300 mg of an antibody described herein (e.g., DX-2930 in doses of 100 mg or 300 mg) is present in a composition optionally containing a pharmaceutically acceptable carrier, for example, a pharmaceutically acceptable composition or a pharmaceutical composition.

[0103] Pharmacopoeia-acceptable carriers include any pharmacologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retarders, and the like. Preferably, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion), but carriers suitable for inhalation and intranasal administration have also been considered.

[0104] A pharmaceutically acceptable salt is one that retains the desired biological activity of a compound without imparting undesirable toxic effects (see, for example, Berge, SM, et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Examples of 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, and phosphorous acid, as well as salts derived from non-toxic organic acids such as fatty mono- and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and fatty and aromatic sulfonic acids. Examples of base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0105] The composition may be in various forms. These include, for example, liquid formulations (e.g., injectable and drip-preservable liquid formulations), dispersants or suspensions, tablets, pills, powders, liposomes, and liquid, semi-solid, and solid dosage forms such as suppositories. This form may depend on the intended mode of administration and therapeutic use. Many compositions are in the form of injectable or drip-preservable liquid formulations, 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, plasma kallikrein-binding protein is administered by intravenous drip or injection. In another preferred embodiment, plasma kallikrein-binding protein is administered by intramuscular or subcutaneous injection. In yet another preferred embodiment, plasma kallikrein-binding protein is administered by intraperitoneal injection.

[0106] As used herein, the terms "parenteral administration" and "administered parenterally" mean, but are not limited to, methods of administration other than enteral and local administration, usually by injection, including intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0107] The composition can be formulated as a liquid, microemulsion, dispersant, liposome, or other indicated structure suitable for high-concentration drugs. Sterile injectable liquids can be prepared by incorporating the required amount of binding protein into a suitable solvent containing one or a combination thereof of the above components, and then sterile filtration as necessary. Generally, dispersants can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components derived from those listed above. For sterile powders for the preparation of sterile injectable liquids, preferred preparation methods are vacuum drying and lyophilization, where the powder of the active component and any additional desired components is collected from their pre-sterile filtered solutions. Appropriate fluidity of the liquid can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, for example, monostearate and gelatin.

[0108] Plasma kallikrein-binding antibodies can be administered by various methods, including intravenous injection or infusion. For example, for use in some therapies, plasma kallikrein-binding proteins are administered at concentrations of approximately 1-100 mg / m². 2 Or 7-25 mg / m² 2The drug can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg / min to reach the desired dose. The route and / or form of administration will vary depending on the desired outcome. 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 microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods are available for preparing such formulations. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., 1978, Marcel Dekker, Inc., New York.

[0109] Pharmaceutical compositions can be administered using medical devices. For example, in one embodiment, the pharmaceutical compositions disclosed herein can be administered using devices such as needleless subcutaneous injection devices, pumps, or implants.

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

[0111] The administration regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, divided doses may be administered over time, or the dose may be proportionally reduced or increased as suggested by the requirements of the treatment situation. In particular, for ease of administration and dose uniformity, it is beneficial to formulate parenteral compositions into unit dosage forms. Unit dosage forms as described herein refer to physically separate units suitable as unit doses for the subject being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specifications of a unit dosage form may depend directly on (a) the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limits of susceptibility in the individual, which are inherent in the techniques for compounding such active compounds for therapeutic purposes.

[0112] The exemplary and non-limiting range for the therapeutically or prophylactically effective dose of the antibodies described herein is 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg. The anti-plasma kallikrein antibody is 1 to 100 mg / m². 2 Or approximately 5-30 mg / m² 2 The drug can be administered, for example, by intravenous infusion, at a rate of less than 30, 20, 10, 5, or 1 mg / min to reach the dose. The dose value may vary depending on the type and severity of the condition to be alleviated. For specific subjects, a specific dose regimen may be adjusted over time at the discretion of the individual and the professional judgment of the person administering or supervising the administration of the composition.

[0113] In some embodiments, the therapeutic or prophylactic effective dose of the antibody described herein (e.g., DX-2930) is 30-400 mg, 30-300 mg, 30-250 mg, 30-200 mg, 30-150 mg, 30-100 mg, 30-50 mg, 50-400 mg, 50-300 mg, 50-250 mg, 50-200 mg, 50-150 mg, 50-100 mg, 1 The effective dose 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 in between. In some embodiments, the therapeutic or prophylactic effective dose is 30-300 mg. In some embodiments, the therapeutic or prophylactic effective dose is 300 mg or more. In some embodiments, the therapeutic or prophylactic effective dose is 400 mg or more. In some embodiments, the therapeutic or prophylactic effective dose is 100–300 mg (100 mg, 150 mg, 200 mg, 250 mg, or 300 mg).

[0114] In some embodiments, the therapeutic or prophylactic effective dose of the 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, the therapeutic or prophylactic effective dose is 30 mg, 100 mg, or 300 mg. In some embodiments, the therapeutic or prophylactic effective dose is 100 mg or 300 mg. In some embodiments, the therapeutic or prophylactic effective dose is 100 mg. In some embodiments, the therapeutic or prophylactic effective dose is 300 mg.

[0115] In some embodiments, an effective therapeutic or prophylactic dose is administered at least two, three, four, five, six, seven, eight, nine, ten, or more times. In some embodiments, an effective therapeutic or prophylactic dose is administered daily, every other day, every three days, every four days, every five days, every six days, weekly, every week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, or more. In some embodiments, an effective therapeutic or prophylactic dose is 100 mg or 300 mg, administered every two weeks or every four weeks. In some embodiments, an effective therapeutic or prophylactic dose is 100 mg, and this amount of antibody is administered every two weeks. In some embodiments, an effective therapeutic or prophylactic dose is 300 mg, and this amount of antibody is administered every two weeks or every four weeks. In some embodiments, an effective therapeutic or prophylactic dose is 300 mg, and this amount of antibody is administered every two weeks. In some embodiments, the therapeutic or prophylactic effective dose is 300 mg, which is administered every four weeks.

[0116] In some embodiments, a therapeutically or prophylactically effective dose is one that maintains a plasma or serum concentration of antibody 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 in maintaining a plasma or serum concentration of 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. Plasma or serum concentrations can be measured using appropriate assays, such as plasma kallikrein assays, such as those described herein, immunobase assays, such as ELISA assays or Western blot assays for determining cleaved kininogen, as described herein, or by mass spectrometry.

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

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

[0119] The informational materials of the kit are not limited to this form. In one embodiment, the informational materials may include information about the compound's production, molecular weight, concentration, expiration date, batch, or production location. In one embodiment, the informational materials relate to the use of antibodies to treat, prevent, or diagnose disorders and conditions, such as diseases or conditions related to plasma kallikrein.

[0120] In one embodiment, the information material may include instructions for administering plasma kallikrein-conjugated antibodies in an appropriate manner for performing the method described herein, for example, in an appropriate dose, dosage form, administration format, or administration schedule (for example, the dose, dosage form, administration schedule, or administration format described herein). In another embodiment, the information material may include instructions for administering plasma kallikrein-conjugated antibodies to appropriate subjects, such as a person having a plasma kallikrein-related disease or condition, or a person at risk thereof. For example, the material may include instructions for administering plasma kallikrein-conjugated proteins to a patient having a disorder or condition described herein, such as a plasma kallikrein-related disease, according to, for example, the administration schedule described herein. The information material in the kit is not limited to this form. Often, the information material, such as instructions, is provided in printed form, but it may be in other formats, such as computer-readable media.

[0121] Plasma kallikrein-conjugated antibodies can be supplied in any form, such as liquid, dry, or lyophilized. Preferably, the plasma kallikrein-conjugated antibody is substantially pure and / or sterile. If the plasma kallikrein-conjugated antibody is supplied in a liquid formulation, the formulation is preferably an aqueous solution, and a sterile formulation is preferred. If the plasma kallikrein-conjugated antibody is supplied in a dry form, it is generally reconstituted by adding a suitable solvent. A solvent, such as sterile water or buffer, can optionally be provided in the kit.

[0122] The kit may comprise one or more containers for a composition containing a plasma kallikrein-conjugated antibody. In some embodiments, the kit comprises separate containers, dividers, or compartments for the composition and informational materials. For example, the composition may be contained in a bottle, vial, or syringe, and the informational materials may be contained in association with the container. In other embodiments, the separate components of the kit are contained in a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe with informational materials in the form of labels attached. In some embodiments, the kit comprises multiple (e.g., one pack) individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the plasma kallikrein-conjugated antibody. For example, the kit comprises multiple syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of plasma kallikrein-conjugated antibody. The containers of the kit may be airtight, water-resistant (e.g., impermeable to changes in moisture or evaporation), and / or light-shielding.

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

[0124] treatment In some embodiments, this disclosure provides the use of antibodies that bind to active plasma kallikrein (e.g., human active plasma kallikrein) in the treatment of HAE. Antibodies suitable for use in the therapies described herein include DX-2930 or its functional variants as described herein, antibodies that bind to the same epitopes 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," is a C1 esterase inhibitor deficiency, or C1 inhibitor deficiency, hereditary angioedema (HANE). HAE is characterized by recurrent episodes of severe swelling (edema) that can affect, for example, the limbs, face, genitals, gastrointestinal tract, and airways. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction that may include throat swelling and sudden hoarseness; recurrent episodes of abnormal abdominal colic with no apparent cause; and / or intestinal swelling, which can be severe and may cause abdominal colic, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with HAE develop a non-itchy rash called erythema annulare between attacks.

[0126] Airway swelling can be life-threatening, and some patients die. The mortality rate is estimated to be between 15% and 33%. HAEs account for approximately 15,000 to 30,000 emergency outpatient visits per year.

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

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

[0129] HAE is inherited in an autosomal dominant pattern, meaning that an affected person may inherit the mutation from one affected parent. Novel mutations in the gene can also occur, and therefore HAE can occur even in people with no family history of the disease. It is estimated that 20-25% of cases result from novel, spontaneous mutations.

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

[0131] Mutations in the F12 gene have been linked to several cases of hereditary angioedema type III. The F12 gene provides instructions for the production of coagulation factor XII. In addition to its crucial role in blood clotting, factor XII is also an important inflammatory stimulant and is involved in bradykinin production. Certain mutations in F12 result in the production of increasedly active factor XII. As a result, more bradykinin is produced, making the blood vessel walls more leaky, which leads to the development of swelling. The causes of other cases of hereditary angioedema type III are still unknown. Mutations in one or more unidentified genes may be responsible for the impairment in these cases.

[0132] HAE can exist similarly to other forms of edema caused by allergies or other medical conditions, but its causes and treatments are significantly different. When hereditary angioedema is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, but these are generally ineffective for HAE. However, epinephrine may be used in life-threatening reactions. Misdiagnosis also leads to unnecessary surgical exploration in patients with abdominal swelling, and in some HAE patients, abdominal pain has been inappropriately misdiagnosed as psychosomatic.

[0133] Treatment of C1 inhibitors and other treatments for HAE are described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0134] Acute treatment for HAE attacks is provided to stop the progression of edema as quickly as possible. One acute treatment is a C1 inhibitor concentrate from donor blood administered intravenously. 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 inhibitors, can be used as an alternative.

[0135] C1 inhibitors purified from human blood have 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 prevention. Rhucin (Pharming) is a recombinant C1 inhibitor under development that does not pose a risk of transmission of infectious diseases from pathogens of human blood origin.

[0136] Treatment for acute HAE attacks may also include drug therapy for pain relief and / or intravenous infusion.

[0137] Other modes of treatment can stimulate the synthesis of C1 inhibitors or reduce their consumption. Androgenic drug therapies, such as danazol, can reduce the frequency and severity of seizures by stimulating the production of C1 inhibitors.

[0138] Helicobacter pylori can trigger abdominal cramps. Antibiotics used to treat Helicobacter pylori can reduce abdominal cramps.

[0139] Newer treatments target the cascades they interact with. Ecalantide (KALBITOR®, DX-88, Dyax) inhibits plasma kallikrein and is approved in the United States. Icatibant (FIRAZYR®, Shire) inhibits bradykinin B2 receptors and is approved in Europe and the United States.

[0140] The diagnosis of HAE may depend, for example, on family history and blood tests. Findings from studies related to types I, II, and III of HAE 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, the symptoms of HAE can be assessed using questionnaires, such as those completed by the patient, clinician, or family member. Such questionnaires are known to those skilled in the art and include, for example, the Visual Analog Scale (VAS). See, for example, McMillan, CV et al. Patient. 2012;5(2):113-26.

[0142] Treatment of HAE with anti-PKal antibodies This disclosure provides a method for treating hereditary angioedema (HAE) (for example, alleviating, stabilizing, or eliminating one or more of its symptoms) by administering the antibodies described herein (for example, a therapeutically effective dose of the antibodies described herein) to subjects who have or are suspected of having HAE, for example, according to the administration schedule described herein. Furthermore, this disclosure provides a method for treating HAE by administering the antibodies described herein (for example, a therapeutically effective dose of the antibodies described herein) for example, according to the administration schedule described herein, or in combination with a second treatment, such as one other agent described herein. This disclosure also provides a method for preventing HAE or its symptoms by administering the antibodies described herein (for example, a prophylactically effective dose of the antibodies described herein) to subjects at risk of developing HAE (for example, subjects with a family history of HAE or with a genetic predisposition to HAE), for example, according to the administration schedule described herein. In some examples, the subjects may be human patients who do not have symptoms of HAE at the time of treatment.

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

[0144] Methods of administering kallikrein-binding antibodies and other drugs 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 specific drug used. For example, antibodies can be used as competitors to inhibit or reduce undesirable interactions between plasma kallikrein and its substrates (e.g., factor XII or HMWK). The antibody dose may be sufficient to inhibit 90%, 95%, 99%, or 99% of the activity of plasma kallikrein in the patient, particularly at the site of disease. This may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg / Kg. For IgG with a molecular weight of 150,000 g / mol (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM binding sites in a 5 L blood volume.

[0145] In one embodiment, the antibody is used, for example, to inhibit the activity of plasma kallikreins in vivo (e.g., to inhibit at least one plasma kallikrein, such as the production of factor XIIa and / or bradykinin). The binding protein can be used on its own or to bind to drugs such as cytotoxic drugs, cytotoxic enzymes, or radioisotopes.

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

[0147] The method of administering plasma kallikrein-conjugated antibodies is described in "Pharmaceutical Compositions." The appropriate dose of the molecule used depends on the age and weight of the subject, as well as the specific drug used. The antibody can be used, for example, as a competing agent to inhibit or reduce undesirable interactions between natural or pathological drugs and plasma kallikrein.

[0148] A therapeutically effective dose of the antibodies described herein can be administered to subjects who have, are suspected of having, or are at risk of having HAE to treat the disorder (e.g., alleviate or improve the symptoms or characteristics of the disorder, delay, stabilize, and / or halt the progression of the disease).

[0149] The antibodies described herein can be administered in therapeutically effective doses. A therapeutically effective dose of an antibody is an amount that is effective in treating a subject, for example, by curing, alleviating, reducing or improving at least one symptom of a disorder in the subject to a degree greater than that predicted in the absence of such treatment, when administered to the subject in a single or multiple dose.

[0150] The dose regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or the dose may be administered in several divided doses over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation. For ease of administration and dose uniformity, it is particularly preferable to formulate the parenteral composition in unit dosage forms. As used herein, unit dosage forms refer to physically separate units suitable as unit dosage forms for the target to be treated; each unit contains a predetermined amount of the active compound calculated to produce a desired therapeutic effect in conjunction with the required pharmaceutically acceptable carrier.

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

[0152] In some embodiments, the patient can be monitored before, during, or after treatment for adverse effects (e.g., elevated creatine phosphatase levels) and / or the level of pKal inhibition by the antibody (e.g., serum or plasma antibody concentration, or pKal activity level). If adverse effects are observed, the antibody dose may be reduced or treatment may be discontinued. If the inhibition level is below the minimum therapeutic level, a further 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 the initial dose) to evaluate the therapeutic effect. If the plasma or serum concentration of the antibody is less than 80 nM, follow-up doses are 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 a subject, for example, by an immunoassay or MS assay. Alternatively, the plasma or serum concentration of the antibody may 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 for measuring cleaved kininogen as described herein.

[0154] Alternatively, the plasma or serum levels of creatine kinase can be monitored during the course of treatment. If elevated levels of creatine kinase are found during treatment, the antibody dose may be reduced or treatment may be discontinued.

[0155] In some embodiments, the optimal dose (e.g., optimal prophylactic dose or optimal therapeutic dose) of the anti-pKal antibody described herein (e.g., DX-2930 or its antigen-binding fragment) can be determined as follows: An initial dose of the antibody is administered to the 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 antibody dose is increased in subsequent doses. The antibody dose that maintains a plasma concentration of the antibody greater than approximately 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 further adjusted based on creatine phosphokinase levels; for example, if an increase in creatine phosphokinase is observed during treatment, the antibody dose can be reduced.

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

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

[0158] The plasma kallikrein-conjugated antibodies described herein can be administered in combination with one or more current therapies for treating HAE. For example, DX-2930 or its functional variants described herein can be co-administered with a second anti-HAE agent such as ecalantide, 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 drugs or treatments to treat the same patient, where the use or action of the drugs or treatments overlaps over time. Drugs or treatments may 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 at different times. The time between the administration of one drug and the administration of another may be in minutes, hours, days, or weeks. The use of plasma kallikrein-conjugated antibodies as described herein may also be used to reduce the dose of another treatment, for example, to reduce side effects associated with the other drug being administered. Therefore, combination may include administering the second drug at a dose at least 10, 20, 30, or 50% lower than that used in the absence of plasma kallikrein-conjugated antibodies.

[0160] Combination therapy may include administering drugs that reduce the side effects of other treatments. These drugs may be drugs that reduce the side effects of treatment for plasma kallikrein-related diseases.

[0161] Without further detail, those skilled in the art will likely be able to fully utilize the present invention based on the above description. Therefore, the following specific embodiments are constructed merely as examples and do not in any way limit the remainder of this disclosure. Publications cited herein are incorporated by reference for the purposes or subjects of this specification.

[0162] Examples Example 1: A single increasing dose trial of DX-2930 in healthy volunteers. A single increasing dose was administered to healthy volunteers using the following doses of DX-2930: 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Data from each volunteer were 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. Here, the signal sequence is italicized. The CDR is in bold and underlined. [ka] [ka] [ka] [ka] [Table 1]

[0163] Phase 1a test design This trial was randomized, double-blind, and placebo-controlled. Single and increasing doses of DX-2930 were administered subcutaneously to healthy subjects. Participants were randomly assigned to cohorts of 1–4 subjects corresponding to a single dose (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg). Each cohort included 6 subjects treated with the active drug and 2 subjects treated with placebo. After completion of the dose schedule, all subjects were monitored for 16 weeks.

[0164] Safety results DX-2930 was well-tolerated at a maximum single dose of 3.0 mg / kg without dose-limiting toxicity. Therefore, this study did not collect evidence of any clinically significant safety signals associated with DX-2930.

[0165] Clinical study results showed no clinical imbalance between DX-2930 and placebo regarding any adverse events. The most commonly reported adverse event was headache (25% of patients treated with DX-2930 and 25% of patients treated with placebo). There were no serious adverse events, and all adverse events resolved.

[0166] Analysis of vital signs, physical tests, and electrocardiogram (ECG) revealed an upper respiratory tract infection in one patient treated with a dose of 0.1 mg / kg. However, the researchers reported that this infection was moderate and not treatment-related. Otherwise, no abnormalities were observed in the vital signs, physical tests, or ECG of the patient.

[0167] The testing of anti-drug antibodies did not collect evidence of antibody seroconversion.

[0168] Only two subjects with severe adverse events were reported by blinded researchers as being treatment-related. An elevated creatine phosphokinase level of 902 U / L (reference range: 21–215 U / L) was observed in subjects treated with 0.1 mg / kg of DX-2930 (4.2% of all subjects treated with DX-2930). An elevated creatine phosphokinase level of 1967 U / L (reference range: 32–294 U / L) was observed in subjects treated with placebo (12.5% ​​of placebo-treated subjects). The results did not indicate any lab abnormalities or clinical significance associated with any other adverse events. No injection site reactions occurred in any of the subjects. [Table 2] *Treatment-related adverse events (AEs): The association of adverse events with the investigational drug was evaluated by blinded researchers. Note: Here, the term "adverse event" (AE) specifically refers to adverse events that occur as a result of treatment. An adverse event was considered treatment-related if its onset occurred between the time of administration of the study drug and the final follow-up visit at 112 days post-treatment, or, in cases where the onset time preceded the administration of the study drug, if the AE increased in severity during the 112-day post-treatment follow-up period.

[0169] Pharmacodynamic (PD) and pharmacokinetic (PK) results Table 3 provides an evaluation of pharmacokinetic parameters for each dose cohort. Mean C max and AUC last The values ​​exhibit a precise linear dose-dependence consistent with antibodies of good quality. Drugs with long half-lives allow for infrequent dosing schedules to achieve stable, consistent blood levels. DX-2930 showed a consistent extension of nearly three weeks in half-life across all dose groups.

[0170] Therapeutic candidates for the prevention of HAEs should have long half-lives and predictable pharmacokinetic profiles to enable low-frequency administration and logical technical dosing principles. DX-2930 provides a consistent pharmacokinetic profile, thereby enabling the determination of dosing regimens that provide significant therapeutic benefits to HAE patients. [Table 3]

[0171] The pharmacokinetic (PK) parameters of DX-2930 were evaluated after a single dose in healthy subjects. Following a single dose of 3 mg / kg, plasma drug concentrations exceeding the target level of 80 nM were achieved. Drug levels around or above 80 nM were maintained for approximately 10 days (Figure 7). Drug levels continued to accumulate with repeated administration until a steady state was reached. Even after a single dose of DX-2930 alone, drug levels exceeding the target 80 nM were achieved and maintained for a long period. The PK data from this study support the feasibility of a dosing strategy to obtain and then maintain the target plasma drug concentration exceeding 80 nM. Furthermore, drug levels higher than 80 nM may be achieved as needed to obtain sufficient plasma kallikrein inhibition associated with HAE prevention.

[0172] Pharmacodynamic (PD) evaluation of DX-2930 was performed. To further characterize DX-2930, exploratory biomarker assays were performed ex vivo on target plasma samples to assess the molecular pharmacodynamic profile. Two independent assays were performed: a plasma kallikrein activity assay using an artificially generated fluorescent agent to measure the cleavage of kininogen, the natural substrate of plasma kallikrein from which bradykinin is derived, and a Western blot assay.

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

[0174] Figure 9 illustrates the pharmacodynamic effects of DX-2930. Plasma from subjects treated with the study drug was activated ex vivo to induce a contact phase pathway, thereby stimulating plasma kallikrein production and activity. pKal activity was measured via a fluorescence assay. The data represent those from the 1 mg / kg and 3 mg / kg groups. Inhibition of plasma kallikrein was particularly evident in the 1 and 3 mg / kg dose groups. No recognizable inhibition was observed in the 0.1 mg / kg or placebo groups. The observed inhibition was both dose- and time-dependent, confirming the inhibitory activity of DX-2930. The PD effect of DX-2930 supports the PK data for DX-2930.

[0175] Therapeutic uses of DX-2930 For effective prevention, a necessary condition for efficacy is that the relevant drug target is continuously inhibited at levels exceeding the minimum required amount. Gaps in the inhibitory range should be minimized or completely avoided. If the inhibition level falls below the minimum required level, the individual is biologically vulnerable to activation of the pathological process and may be at clinical risk regarding disease events.

[0176] There are no widely established principles of prevention for HAEs. In HAEs, plasma kallikrein represents an effective drug target that is important for the cause of edema attacks. Preventing HAE attacks may require continuous maintenance of plasma kallikrein inhibition beyond the minimum therapeutic level. Gaps in the range over time should be minimized to avoid vulnerable periods. This need is further emphasized by the phenomenon of a positive feedback loop, which is hypothesized to play a crucial role in HAE attacks. Once this cascade is initiated, activation of plasma kallikrein leads to activation of factor XII, which then drives the production of more plasma kallikrein.

[0177] The inhibition of plasma kallikrein by DX-2930 and ekalantide, a known therapeutic agent approved in the United States for the acute treatment of HAE attacks, was compared in an in vitro assay. In this system, human plasma was exposed to a drug that initiated the contact phase and converted prekallikrein to active plasma kallikrein.

[0178] The peak plasma concentration of ecalantide 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 has equivalent potency to ecalantide (Figure 5). If 80 nM appears to be an appropriate plasma kallikrein level for HAE attacks, and if the potency of DX-2930 is equivalent to ecalantide in inhibiting plasma kallikrein in this concentration range, then it can be hypothesized that maintaining DX-2930 at a plasma drug concentration continuously above 80 nM would prevent HAE attacks (Figure 6). The target plasma drug level of DX-2930 at 80 nM appears reliable based on currently available data and our understanding of disease biology. While 80 nM is the initial target, lower or higher drug levels of DX-2930 may be necessary to obtain therapeutic doses.

[0179] Example 2: Multiple increasing dose studies 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 shows the predicted plasma concentrations achieved after repeated administration of DX-2930 at a dose of 3 mg / kg. The initial concentration profile is consistent with the profile observed with a single dose in healthy subjects.

[0180] PK modeling can be used to predict the pharmacodynamic and pharmacokinetic properties of DX-2930 after chronic administration. Figure 8 shows 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 a steady state is reached, repeated administration will continuously maintain drug concentrations around or above the initially targeted level of 80 nM (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 administered one week apart. Biomarker data (along with pharmacokinetics) will be evaluated to determine which dose to use in follow-up studies to assess the efficacy of DX-2930 in preventing HAE attacks (at different doses).

[0182] Example 3: Pharmacological modeling of a dosing regimen to investigate the long-term prevention of hereditary angioedema. method In vitro inhibition of pKal by ecalantide and DX-2930 was evaluated using synthetic substrate assays. Modeling was performed using pharmacokinetic (PK) data from a single increasing dose subcutaneous administration study of DX-2930 in healthy subjects.

[0183] result In vitro pKal assays showed that DX-2930 and ecalantide exhibited comparable pharmacodynamic (PD) activity at a concentration of 80 nM. Plasma concentrations of pKal inhibitors below 80 nM presented only partial prophylactic effects, providing further evidence for the moderate and high-dose hypotheses rather than the low-dose hypothesis. Therefore, continuously maintaining drug levels of DX-2930 above approximately 80 nM should reach the pKal inhibition levels explained by the moderate-dose hypothesis. PK modeling indicates that chronic DX-2930 administration results in stable plasma drug concentrations of over 80 nM using 100 mg every two weeks (or 300 mg every four weeks) and over 200 nM using 300 mg every two weeks.

[0184] Other Embodiments All properties disclosed herein may be combined in any combination. Each property disclosed herein may be replaced by an alternative property that serves the same, equivalent, or similar purpose. Thus, unless otherwise clearly stated, each disclosed property is merely an example of an equivalent or similar property in a general form.

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

Claims

1. A pharmaceutical composition characterized by being used in combination with a therapeutic agent in the treatment of hereditary angioedema (HAE), wherein the pharmaceutical composition comprises an antibody containing the heavy chain variable domain sequence of SEQ ID NO: 3 and the light chain variable domain sequence of SEQ ID NO:

4. The aforementioned therapeutic agent is ecalantide, a C1 inhibitor, or icatibant. The antibody is administered in an effective dose of 150 mg or 300 mg every two weeks or every four weeks, and A pharmaceutical composition wherein the C1 inhibitor is a concentrate of C1 inhibitor from donor blood, fresh frozen plasma (FFP), Berinert P (registered trademark), or Cinryze (registered trademark).

2. The aforementioned antibody EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG The heavy chain sequence, and DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The pharmaceutical composition according to claim 1, comprising the light chain sequence.

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

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody is administered subcutaneously and / or the antibody is administered for prophylactic treatment.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject is a human patient who has, is suspected of having, or is at risk of having HAE.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the administration is by subcutaneous injection.

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