EA045920B1 - PLASMA KALLIKREIN INHIBITORS AND THEIR USE FOR TREATMENT OF ACHIEVEMENTS OF HEREDITARY ANGIONEOUROTIC EDEMA - Google Patents

Description

Cross reference to related applications

This application claims priority to the date of U.S. Provisional Patent Application No. 62/266175, filed December 11, 2015, U.S. Provisional Patent Application No. 62/266192, filed December 11, 2015, and U.S. Provisional Patent Application No. 62/395833, filed September 16, 2016. The entire contents of each of these reference applications are incorporated by reference into this application.

Prerequisites for creating the invention

Plasma kallikrein is a serine protease component of the contact system and a potential target for drugs against various inflammatory, cardiovascular, infectious (sepsis) and oncological diseases (Sainz I.M. et al., Thromb Haemost 98, 77-83, 2007). The contact system is activated either by factor XIIa upon exposure to foreign or negatively charged surfaces, or on the surface of endothelial cells through prolyl carboxypeptidases (Sainz I.M. et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein enhances intrinsic coagulation through feedback activation of factor XII and enhances inflammation through the production of the proinflammatory nonapeptide bradykinin. As the primary kininogenase in circulation, plasma kallikrein is largely responsible for the production of bradykinin in the vascular system. Genetic deficiency of C1 inhibitor protein (C1-INH), the main natural inhibitor of plasma kallikrein, results in hereditary angioedema (HAE). Patients with HAE suffer from acute episodes of painful swelling, often caused by unknown triggers (Zuraw B.L. et al., N Engl J Med 359, 1027-1036, 2008).

The essence of the invention

The present invention provides regimens for treating exacerbation of hereditary angioedema (HAE), reducing the frequency of HAE attacks, or blocking exacerbation of HAE using antibodies capable of binding and inhibiting human plasma kallikrein (pKal) in the active form, for example, antibodies that bind to the same epitope as DX-2930 (a.k.a. SHP643), or compete with DX-2930 for binding to active human pKal.

In some aspects, the present disclosure provides methods for treating an exacerbation of hereditary angioedema (HAE) or reducing the frequency of HAE attacks, comprising administering (e.g., subcutaneously) to a subject in need thereof any of the anti-pKal antibodies described herein (e.g., DX- 2930), in single and/or multiple doses. In some embodiments, the antibody is administered to a subject (eg, a human patient) in multiple doses over a first treatment period, for example, 26 weeks. For example, the antibody can be administered at about 300 mg every two weeks, about 300 mg every four weeks, or about 150 mg every four weeks in the first treatment period.

Any of the methods described in the present invention may further include administering the antibody to the subject for a second treatment period (eg, 26 weeks) after the first treatment period. In some embodiments, the first dose of the second treatment period is administered approximately two weeks after the last dose of the first treatment period. In some embodiments, the second treatment period includes multiple doses of the antibody of about 300 mg every two weeks for, for example, 26 weeks.

Alternatively, the methods may further include administering to the subject a single dose of the antibody (eg, about 300 mg) after the first treatment period. After a single dose, one or more doses of the antibody (eg, about 300 mg every 2 weeks) may be administered if an exacerbation of HAE occurs after a single dose. The first dose of subsequent treatment may be administered within one week of the HAE exacerbation. In some embodiments, there is a gap of at least 10 days between administration of a single dose and the first dose of subsequent treatment.

Other aspects of the present disclosure provide methods for treating an exacerbation of hereditary angioedema (HAE) or reducing the frequency of HAE attacks in a subject who has previously received treatment for HAE, comprising (i) administering (e.g., subcutaneously) to the subject in need thereof any of the anti-pKal antibodies described in the present invention (for example, DX-2930), the antibody in a single dose of about 300 mg; and (ii) further administering the antibody to the subject in multiple doses of about 300 mg every two weeks if the subject experiences an attack of HAE after (i). The subject could undergo a first HAE treatment as described in the present invention. For example, the first HAE treatment may be a treatment comprising the same antibody (eg, DX-2930). In some examples, the first treatment for HAE involves administering the antibody in multiple doses of about 150 mg or 300 mg every four weeks or administering the antibody in multiple doses of about 300 mg every two weeks. Alternatively or in addition, the first dose of multiple doses is given to the subject within one week of the HAE exacerbation.

In any of the treatment methods described in the present invention, the subject may be a human patient having HAE or at increased risk of HAE. In some embodiments, the subject is a human patient having HAE type I or type II. For example, a subject may have at least one exacerbation of HAE in the four weeks prior to the first dose of the first treatment period, or at least two exacerbations of HAE in the eight weeks prior to the first dose of the first treatment period.

- 1045920 of the treatment period. In other examples, the subject may be a human patient who has experienced at least two exacerbations of HAE in the year prior to treatment. Alternatively or in addition, the subject is a human patient who has not had prior treatment, for example, at least two weeks prior to the first dose of the first treatment period.

In some embodiments, a subject to be treated with any of the methods described herein, which include the use of any of the anti-pKal antibodies also described herein (e.g., DX-2930), has received one or more HAE treatments prior to the first dose of anti-pKal antibodies. -pKal antibodies. Such prior HAE treatments may include a C1 inhibitor (eg, C1-INH), a plasma kallikrein inhibitor (eg, ecallantide), a bradykinin receptor antagonist (eg, icatibant), an attenuated androgen (eg, danazol), an antifibrinolytic agent (eg, tranexamic acid), or their combinations. Such a subject could have a period of gradual transition from prior HAE treatment to treatment with the anti-pKal antibody described in the present invention. In some examples, the dosage reduction period may be 2 to 4 weeks. Previous HAE treatment may end either before the first dose of anti-pKal antibody or within three weeks of the subject's first dose of anti-pKal antibody. Alternatively, the subject may be directly switched from any prior HAE treatment to anti-pKal antibody treatment as described herein.

In some embodiments, the subject does not have long-term HAE prophylaxis or HAE treatment, including an angiotensin-converting enzyme (ACE) inhibitor, estrogen-containing drug, or androgen, before the first treatment period, during the first treatment period, and/or during the second treatment period.

The anti-pKal antibody for use in the treatment regimens described in the present invention can be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, the antibody may include the same CDRs as DX-2930. In one example, the antibody is DX-2930.

In any of the methods described in the present invention, the antibody can be formulated into a pharmaceutical composition including a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes sodium phosphate, citric acid, histidine, sodium chloride, and Tween 80. In one example, sodium phosphate is present at a concentration of about 30 mM, citric acid is present at a concentration of about 19 mM, histidine is present at a concentration of about 50 mM, sodium chloride is present at a concentration of about 90 mM and/or polysorbate 80 is about 0.01%.

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

Brief description of drawings:

Fig. 1 shows an exemplary dosing regimen including a first treatment period in which patients are administered DX-2930 at a dose of 300 mg every two weeks, 300 mg every four weeks, or 150 mg every 4 weeks.

Fig. 2 shows an exemplary dosage regimen including a second treatment period following the first treatment period. In the second treatment period, patients are given a single dose of DX-2930 300 mg. If the patient experiences flare-ups of HAE, the antibody is subsequently administered at a dose of 300 mg every two weeks.

Fig. 3 shows 2-chain high molecular weight kininogen (HMWK) in HAE plasma samples from patients treated with DX-2930 on the indicated days at the indicated doses (30 mg, 100 mg, 300 mg, or 400 mg) as detected by Western blot analysis. The baseline level of 2-stranded HMWK found in healthy subjects is indicated by the dotted line. For each day, the columns correspond, from left to right, placebo, 30 mg, 100 mg, 300 mg, and 400 mg.

Detailed Description Definitions

For convenience, before the following description of the present invention, certain terms used in the description, examples, and appended claims are defined. Other terms are defined as they appear in the description.

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

In the context of the present invention, the term about refers to a specific value of ±5%. For example, an antibody of about 300 mg includes any amount of antibody between 285 mg-315 mg.

The term antibody refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. An antibody may include at least one heavy (H) chain that includes an immunoglobulin heavy chain variable domain (VH), at least one light chain that includes an immunoglobulin light chain variable domain (VL), or both. For example, an antibody may include a variable

- 2 045920 heavy (H) chain region (abbreviated in the present invention as VH or HV) and light chain variable region (L) (abbreviated in the present invention as V _L or LV). In another example, the antibody includes two heavy (H) chain variable regions and two light chain (L) variable regions.

In the context of the present invention, the term antibody covers not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also their antigen binding fragments (such as Fab, Fab', F(ab') ₂ , Fv), single chain (scFv) domain (dAb) antibody fragments (de Wildt et. al., Euro. J. Immunol. (1996) 26(3): 629-639), any mutants thereof, fusion proteins containing part of the antibody, humanized antibodies, chimeric antibodies, diabodies , linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. An antibody includes any class of antibody such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant domain of the heavy chains of antibodies, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Antibodies can be from any source, but primates (human and non-human primates) and primatized antibodies are preferred.

The VH and/or _VL regions may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other changes. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can bind to another immunoglobulin variable domain sequence to form an antigen binding site, for example, a structure that preferentially interacts with plasma kallikrein.

The VH and/or _VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). The dimensions of the framework region and CDR have been determined (see, Rabat, E. A., 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 definitions are used in the present invention. Each VH and VL typically consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In addition to the VH or _VL regions, the heavy chain or light chain of an antibody may include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, wherein the immunoglobulin heavy and light chains are linked together, for example, by disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2 and CH3. The light chain constant region includes the CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with the antigen. Antibody constant regions typically mediate binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. Immunoglobulin light chains can be kappa or lambda type. In one embodiment, the antibody is glycosylated. The antibody may be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of the antibody may be human or substantially human. For example, one or more variable regions may be human or substantially human. For example, one or more of the CDRs may be human, for example, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and/or LC CDR3. Each of the light chain (LC) and/or heavy chain (HC) CDRs can be human. The CDR3 HC may be human. One or more framework regions may be human, for example, FR1, FR2, FR3 and/or FR4 in the HC and/or LC. For example, the Fc region may be human. In one embodiment, all framework regions are human, eg, derived from a human somatic cell, such as a hematopoietic cell that produces immunoglobulins, or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, for example, encoded by a germline nucleic acid. In one embodiment, implementing

- 3 045920 framework (FR) residues of a selected Fab can be converted to the amino acid type of the corresponding residue in the most related primate germline gene, especially in a human germline gene. One or more constant regions may be human or substantially human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of the immunoglobulin variable domain, constant region, constant domains (CH1, CH2, CH3 and/or CL1) or the antibody as a whole may be human or essentially human.

The antibody may be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as many immunoglobulin variable region genes. Full-length immunoglobulin light chains (about 25 KDa or about 214 amino acids) are encoded by the variable region gene at the II2 end (about 110 amino acids) and the kappa or lambda constant region gene at the COOH end. Full-length immunoglobulin heavy chains (about 50 KDa or about 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and one of the other constant region genes mentioned above, such as gamma (encoding about 330 amino acids). The length of human NS varies considerably, with CDR3 NS ranging from approximately 3 amino acid residues to over 35 amino acid residues.

The term antigen-binding fragment of a full-length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed by the term antigen binding fragment of a full-length antibody and which retain functionality include (i) Fab fragment, a monovalent fragment consisting of VL, _VH , CL and CH1 domains; (ii) F(ab')2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of _VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and _VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) a dedicated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and _VH , are encoded by different genes, they can be combined using recombinant methods with a synthetic linker, allowing them to be produced as a separate protein chain in which the VL and VH regions are paired to form monovalent molecules known as single-chain Fv (scFv). See, for example, US Pat. Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibody fragments can be obtained using any suitable method, including conventional methods known to those skilled in the art.

The term monospecific antibody refers to an antibody that exhibits a single binding specificity and affinity for a particular target, such as an epitope. This term includes a monoclonal antibody or monoclonal antibody composition, which, in the context of the present invention, refers to a preparation of antibodies or fragments thereof with a single molecular composition, regardless of how the antibody was formed. Antibodies are modified at the germline gene level by reverting one or more non-germline amino acids in the framework regions to the corresponding germline antibody amino acids, provided that binding properties are substantially retained.

The inhibition constant (Ki) is an indicator of inhibitory activity; it represents the concentration of inhibitor required to reduce enzyme activity by half and is independent of enzyme or substrate concentrations. Apparent Ki (K _ijapp ) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of inhibitor (eg, inhibitory binding protein) on the extent of the reaction (eg, enzymatic activity); Fitting the change in value of the pseudo-first order constant as a function of inhibitor concentration to the Morrison equation (Equation 1) yields the apparent Ki value. The Ki value is obtained from the Y-intercept obtained from linear regression analysis of a plot of K _ijapp versus substrate concentration.

( (% +Ι + Ε)-/Κ _ν№ +Ι + Ε) ² -4·Ι·Ε] v = v„ - v _n ----------------- -----------------------° ° 2-E

Equation 1

Where v=measured speed; v0=velocity in the absence of inhibitor; K _Lapp apparent inhibition constant; [total inhibitor concentration; and E=total enzyme concentration.

In the context of the present invention, the term binding affinity refers to the apparent association constant or K _A . K _A is the reciprocal of the dissociation constant (K _D ). A binding antibody, for example, may have a binding affinity of at least 105, 106, 107, 108, 109, 1010 and 1011 M-1 for a particular target molecule, for example plasma kallikrein. The higher binding affinity of the binding antibody to the first target compared to the second target may be indicated by a higher KA value (or lower numerical KD value) for binding

- 4 045920 binding of the first target than the K _A value (or the numerical value of K _D ) for binding the second target. In such cases, the binding antibody has specificity for a first target (eg, a protein in a first conformation or a mimic thereof) over a second target (eg, the same protein in a second conformation or a mimic thereof; or a second protein). Differences in binding affinity (eg for specificity or other comparisons) may be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 70, 80, 90, 100 , 500, 1000, 10,000 or 10 ⁵ times.

Binding affinity can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (eg, using fluorescence analysis). Exemplary conditions for assessing binding affinity include the use of HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) surfactant P20). These methods can be used to measure the concentration of bound and free binding protein as a function of protein (or target) concentration. The concentration of the bound binding protein ([Bound]) is related to the concentration of the free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target, where (N) denotes the number of binding sites per target molecule, through the following equation:

[Bound]=N [Free]/((1/K _a )+[Free]).

An accurate determination of K _a is not always necessary, although since it is sometimes sufficient to obtain a quantitative measure of affinity, for example determined using a method such as ELISA or FACS analysis, it is proportional to K _a and can therefore be used for comparisons such as determination whether the affinity is higher, eg 2-fold higher, to obtain a qualitative measure of affinity or to infer affinity, eg from activity in a functional assay, eg in vitro or in vivo assay.

The term binding antibody (or binding protein, used interchangeably in the present invention) refers to an antibody that can interact with a target molecule. The term is used interchangeably with ligand. Plasma kallikrein-binding antibody refers to an antibody that can interact with (eg, bind to) plasma kallikrein, and includes, but is not limited to, antibodies that preferentially or specifically interact with and/or inhibit plasma kallikrein. An antibody inhibits plasma kallikrein if it causes a decrease in plasma kallikrein activity compared to plasma kallikrein activity in the absence of the antibody and under the same conditions.

A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine).

It is possible that one or more amino acid residues of the framework region and/or CDR of the binding protein may include one or more mutations (e.g., substitutions (eg, conservative or non-essential amino acid substitutions), insertions or deletions) relative to the binding protein described in the present invention. The plasma kallikrein binding protein may have mutations (e.g., substitutions (e.g., conservative or non-essential amino acid substitutions), insertions, or deletions) (e.g., at least one, two, three, or four and/or less than 15 , 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations) relative to the binding protein described in the present invention, for example, mutations that do not significantly affect the function of the protein. Mutations may be present in framework regions, CDRs and/or constant regions. In some embodiments, the mutations are present in the framework region. In some embodiments, mutations are present in the CDR. In some embodiments, the mutations are present in the constant region. Whether a particular replacement is portable or not, i.e. whether there will be an adverse effect on biological properties such as binding activity can be predicted, for example, by assessing whether the mutation is conserved, or using the method of Bowie, et al. (1990) Science 247:1306-1310

Essentially, a human immunoglobulin variable region is an immunoglobulin variable region comprising a sufficient number of human amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An essentially human antibody is an antibody comprising a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.

An epitope refers to a site on a target compound that is bound by a binding protein (eg, an antibody such as a Fab or full-length antibody). If the target compound

- 5 045920 is a protein, the site may consist entirely of amino acid components, consist entirely of chemical modifications of the amino acids of the protein (for example, glycosyl fragments) or consist of combinations thereof. Overlapping epitopes include at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature.

The first binding antibody binds to the same epitope as the second binding antibody if the first binding antibody binds to the same site on the target compound that the second binding antibody binds to, or binds to a site that overlaps (e.g., 50%, 60% %, 70%, 80%, 90% or 100% overlap, for example, with respect to amino acid sequence or other molecular feature (eg, glycosyl group, phosphate group or sulfate group) with the site to which the second binding antibody binds.

The first binding antibody competes for binding with the second binding antibody if the binding of the first binding antibody to its epitope decreases (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 100% or more) the amount of the second binding antibody that binds to its epitope. Competition may be direct (eg, the first binding antibody binds to an epitope that is the same as or overlaps with the epitope bound by the second binding antibody), or indirect (eg, binding of the first binding antibody to its epitope causes a steric change in target compound that reduces the ability of the second binding antibody to bind its epitope).

Calculations of homology or sequence identity between two sequences (these terms are used interchangeably herein) are performed as follows. Sequences are aligned for purposes of optimal comparison (eg, gaps may be included in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for purposes of comparison). The optimal alignment is determined as the best estimate using the GAP program in the GCG software package with a Blossum 62 substitution matrix, with a single gap penalty of 12, a gap expansion penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides in the corresponding amino acid positions or nucleotide positions. 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, then the molecules are identical at that position (in the context of the present invention, amino acid or nucleic acid identity is equivalent to amino acid or nucleic acid homology). The percentage identity between two sequences is a function of the number of identical positions shared by the sequences.

In a preferred embodiment, the length of the reference sequence being aligned for comparison purposes 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 at least 70%, 80%, 90%, 92%, 95%, 97%, 98% or 100% of the reference sequence length. For example, the reference sequence may be the length of an immunoglobulin variable domain sequence.

A humanized immunoglobulin variable region is an immunoglobulin variable region modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immune response in a normal human. Descriptions of humanized immunoglobulins include, for example, US Patent Nos. 6,407,213 and 5,693,762.

An isolated antibody refers to an antibody isolated from at least 90% of at least one component of a natural sample from which the isolated antibody can be produced. Antibodies may have at least a certain degree of purity if the species or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure by weight.

The patient, subject or host (these terms are used interchangeably) to be treated by the method of the present invention may mean a human or a non-human animal. The subject may be a subject who has previously undergone HAE treatment, such as a treatment comprising an antibody described in the present invention. In some embodiments, the subject is a pediatric subject (eg, an infant, child, or adolescent).

The terms prekallikrein and plasma prekallikrein are used interchangeably in the present invention and refer to the zymogenic form of active plasma kallikrein, also known as prekallikrein.

In the context of the present invention, the term substantially identical (or substantially homologous) is used to designate a first amino acid or nucleic acid sequence containing a sufficient number of identical or equivalent (e.g., similar side chain, e.g., conservative amino acid substitutions) amino acid residues or nucleotides with the second amino acid or nucleic acid sequence, such that the first and second amino acid or nucleic acid sequences have (or

- 6 045920 differentiate proteins having similar activities, for example binding activity, binding preference or biological activity. In the case of antibodies, the second antibody has the same specificity and has at least 50%, at least 25%, or at least 10% affinity for the same antigen.

Sequences similar or homologous (eg, at least about 85% sequence identity) to sequences disclosed in the present invention are also part of the present invention. 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, the plasma kallikrein binding antibody may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with antibody described in the present invention. In some embodiments, the plasma kallikrein binding antibody may have a sequence identity of about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more in framework regions of the HC and/or LC (eg, FR 1, 2, 3 and/or 4 of the HC and/or LC) with an antibody described in the present invention (eg, DX-2930). In some embodiments, the plasma kallikrein binding antibody may have a sequence identity of about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more in CDR HC and/or LC (eg, CDR1, 2 and/or 3 HC and/or LC) with an antibody described in the present invention (eg, DX-2930). In some embodiments, the plasma kallikrein binding antibody may have a sequence identity of about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more in constant region (eg, CH1, CH2, CH3 and/or CL1) with an antibody described in the present invention (eg, DX-2930).

In addition, there is significant identity when nucleic acid segments hybridize under selective hybridization conditions (eg, very stringent hybridization conditions) with the complement of the strand. The nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form.

Statistical significance can be determined by any method known in the art. Examples of statistical tests include: Student's t test, nonparametric Mann-Whitney U test, and nonparametric Wilcoxon test. Some statistically significant relationships have a P value less than 0.05 or 0.02. Particular binding proteins may exhibit a difference, for example in specificity or binding, that is statistically significant (eg, P value <0.05 or 0.02). The terms induce, inhibit, potentiate, enhance, increase, decrease, or the like, for example, indicating noticeable qualitative or quantitative differences between two conditions, can refer to differences, for example, statistically significant differences between two conditions.

A therapeutically effective dose preferably modulates a measurable parameter, e.g., plasma kallikrein activity, to a statistically significant extent by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even more preferably at least about 80% relative to untreated subjects. The ability of a compound to modulate a measurable parameter, eg, a disease-associated parameter, can be assessed in an animal model system predictive of effectiveness in human disorders and conditions. Alternatively, this property of the composition can be assessed by testing the ability of the compound to modulate the parameter in vitro.

The term treatment as used in the present invention refers to the use or administration of a composition comprising one or more active agents to a subject having an allergic disease, a symptom of an allergic disease, or a predisposition to an allergic disease, for the purpose of recovery, healing, relief, mitigation, modification, cure , improvement, health improvement or effect on a disease, symptoms of a disease or predisposition to a disease. Prophylactic treatment, also known as preventative treatment, refers to treatment aimed at protecting an individual or reducing the risk of a disease to which he or she is or may be exposed. In some embodiments, the treatment methods described herein are aimed at preventing the occurrence and/or recurrence of HAE.

The term "prevention of a disease in a subject" refers to pharmaceutically treating a subject, eg, administering a drug, so as to prevent at least one symptom of a disease, i.e. administration occurs prior to the clinical manifestation of an undesirable condition (eg, a disease or other undesirable condition in the host animal) to protect the host from the development of the undesirable condition. Prevention of disease can also be called prophylaxis or prophylactic treatment.

A prophylactically effective amount refers to an amount effective, at the required dosages and for the required period of time, to achieve the desired prophylactic result. In general, since the prophylactic dose is used in subjects before the development of the disease or at an early stage thereof, the prophylactically effective amount will be less than the tera

- 7 045920 pharmaceutically effective amount.

Plasma kallikrein binding antibodies (pKal)

Antibodies that bind plasma kallikrein (anti-pKal antibodies) for use in the methods presented in the present invention can be full-length (for example, IgG (including IgG1, IgG2, IgG3, IgG4), IgM, IgA (including IgA1, IgA2) , IgD and IgE) or may include only an antigen-binding fragment (for example, a Fab, F(ab') ₂ or scFv fragment. The binding antibody may include two immunoglobulin heavy chains and two immunoglobulin light chains or may be a single chain antibody. Kallikrein-binding antibodies plasma, may be recombinant proteins, such as humanized, CDR-grafted, chimeric, deimmunized or in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.In one embodiment, a plasma kallikrein binding antibody , is a monoclonal antibody.

In one aspect, the present invention provides an antibody (eg, an isolated antibody) that binds to plasma kallikrein (eg, human plasma kallikrein and/or mouse kallikrein) and includes at least one immunoglobulin variable region. For example, the antibody includes an immunoglobulin heavy chain (HC) variable domain sequence and/or an immunoglobulin light chain (LC) variable domain sequence. In one embodiment, the antibody binds to and inhibits plasma kallikrein, for example, human plasma kallikrein and/or murine kallikrein.

The antibody may include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the immunoglobulin HC variable domain sequence includes one or more (for example, 1, 2 or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDR of the HC variable domain described in the present invention; (c) the immunoglobulin LC variable domain sequence includes one or more (for example, 1, 2 or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDR of the LC variable domain described in the present invention; (d) the sequence of the immunoglobulin LC variable domain is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the LC variable domain described in the present invention (for example, in general or in wireframe areas or CDRs); (f) the sequence of the immunoglobulin HC variable domain is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the HC variable domain described in the present invention (for example, in general or in wireframe areas or CDRs); (f) the antibody binds to an epitope bound by an antibody described in the present invention or competes for binding with an antibody described in the present invention; (g) primate CDR or primate framework region; (h) the immunoglobulin HC variable domain sequence includes a CDR1 differing in at least one amino acid, but not more than 2 or 3 amino acids, from the HC variable domain CDR1 described in the present invention; (i) the immunoglobulin HC variable domain sequence includes a CDR2 that differs in at least one amino acid, but not more than 2, 3, 4, 5, 6, 7 or 8 amino acids from the HC variable domain CDR2 described in the present invention; (j) the immunoglobulin HC variable domain sequence includes a CDR3 that differs by at least one amino acid, but not more than 2, 3, 4, 5 or 6 amino acids from the HC variable domain CDR3 described in the present invention; (k) the immunoglobulin LC variable domain sequence includes a CDR1 that differs in at least one amino acid, but not more than 2, 3, 4 or 5 amino acids from the LC variable domain CDR1 described in the present invention; (l) the immunoglobulin LC variable domain sequence includes a CDR2 that differs in at least one amino acid, but not more than 2, 3 or 4 amino acids from the LC variable domain CDR2 described in the present invention; (m) the immunoglobulin LC variable domain sequence includes a CDR3 that differs in at least one amino acid, but not more than 2, 3, 4 or 5 amino acids from the LC variable domain CDR3 described in the present invention; (n) the immunoglobulin LC variable domain sequence differs by at least one amino acid, but not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the LC variable domain described in the present invention (e.g., generally or in wireframe areas or CDRs); and (o) the sequence of the immunoglobulin HC variable domain differs by at least one amino acid, but not more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from the HC variable domain described in the present invention (for example, in whole or in frame regions or CDRs).

The plasma kallikrein binding protein may be an isolated antibody (eg, at least 70, 80, 90, 95, or 99% free from other proteins). In some embodiments, the plasma kallikrein binding antibody or composition thereof is isolated from cleavage fragments of the antibody (e.g., DX-2930) that are inactive or partially active (e.g., bind plasma kallikrein with a Ki, _app 5000 nM or more) relative to the antibody , which binds plasma kallikrein. For example, an antibody that binds plasma kallikrein is at least

- 8 045920 is 70% free from such antibody cleavage fragments; in other embodiments, the binding antibody is at least 80%, at least 90%, at least 95%, at least 99%, or even 100% free of antibody cleavage fragments that are inactive or partially active.

The plasma kallikrein binding antibody may further inhibit plasma kallikrein, such as human plasma kallikrein.

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

In some embodiments, the antibody binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope overlapping the active site of plasma kallikrein.

In some aspects, the antibody binds to the same epitope or competes for binding with an antibody described in the present invention.

The antibody can bind to plasma kallikrein, for example human plasma kallikrein, with binding affinities of at least 10 ⁵ , 10 ⁶ , 10 ⁷ , 108, 109, 10 ¹⁰ and 10 ¹¹ M ^-1 . In one embodiment, the antibody binds to human plasma kallikrein with Koff slower than 1x10 ^-3 , 5x10 ^-4 sec ^-1 or 1x10 ^-4 sec ^-1 . In one embodiment, the antibody binds to human plasma kallikrein with Kon faster than 1x10 ² , 1x10 ³ or 5x10 ³ M ^-1 sec ^-1 . In one embodiment, the antibody binds to plasma kallikrein but does not bind to tissue kallikrein and/or plasma prekallikrein (e.g., the antibody binds to tissue kallikrein and/or plasma prekallikrein less efficiently (e.g., 5, 10, 50, 100, or 1000 times binds less or not at all, for example, compared to a negative control) than it binds to plasma kallikrein.

In one embodiment, the antibody inhibits human plasma kallikrein activity, for example, with a Ki of less than 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 and 10 ^-10 M. The antibody may have, for example, an IC ₅₀ less than 100 nM, 10 nM, 1, 0.5 or 0.2 nM. For example, the antibody may modulate plasma kallikrein activity, as well as the production of factor XIIa (eg, from factor XII) and/or bradykinin (eg, from high molecular weight kininogen (HMWK)). The antibody may inhibit plasma kallikrein activity and/or the production of factor XIIa (eg, from factor XII) and/or bradykinin (eg, from high molecular weight kininogen (HMWK)). The affinity of an antibody for human plasma kallikrein may be characterized by a KD of less than 100 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. In one embodiment, the antibody inhibits plasma kallikrein but does not inhibit tissue kallikrein (e.g., the antibody inhibits tissue kallikrein less effectively (e.g., 5, 10, 50, 100, or 1000 times less or no inhibition, e.g., compared to negative control) than it inhibits plasma kallikrein).

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

Antibodies that bind plasma kallikrein may have HC and LC variable domain sequences included in a single polypeptide (eg, scFv) or different polypeptides (eg, IgG or Fab).

In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another aspect, 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 embodiments, the antibody comprises a Fab2', scFv, minibody, scFv::Fc fusion protein, Fab::HSA fusion protein, HSA::Fab fusion protein, Fab::HSA::Fab fusion protein, or other molecule that includes an antigen-binding site of one of the binding proteins described. The VH and VL regions of these Fabs can be represented 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 design.

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

In one embodiment, the antibody is a primate antibody or a primatized antibody or is non-immunogenic in humans. For example, the antibody includes one or more primate antibody framework regions, e.g., all primate framework regions or framework regions

- 9 045920 ti, at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the framework areas of the primate. In one embodiment, the antibody comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain. The term primate includes humans (Homo sapiens), chimpanzees (Pal troglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, the Madagascan monkey (Daubentonia madagascariensis) and tarsiers.

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

In some embodiments, the antibody does not include sequences from mice or rabbits (eg, is not a mouse or rabbit antibody).

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

In one example, a functional variant of DX-2930 includes the same complementarity determining regions (CDRs) as DX-2930. In another example, functional variants of DX-2930 may contain one or more mutations (eg, conservative substitutions) in FR V _H or VL compared to the VH and VL of DX-2930. Preferably, such mutations do not occur at residues that are predicted to interact with one or more CDRs that can be determined by conventional methods. In other embodiments, the functional variants described in the present invention contain one or more mutations (eg, 1, 2, or 3) in one or more CDR regions of DX-2930. Preferably, such functional variants retain the same antigen binding regions/residues as the parent antibody. In other embodiments, a functional variant of DX-2930 may include a V _H chain that comprises at least 85% of the amino acid sequence (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the _VH sequence of DX-2930, and/or a VL chain that is at least 85% identical in amino acid sequence (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98 % or 99%) identical to the sequence of VL DX-2930. These variants are capable of binding to the active form of plasma kallikrein and preferably do not bind to prekallikrein.

The percentage identity of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is included in the NBLAST and XBLAST programs (version 2.0) 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 protein molecules of interest. If gaps exist 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, you can use the default parameters of the corresponding programs (for example, XBLAST and NBLAST).

In some embodiments, the antibody used in the methods and compositions provided herein may be a DX-2930 antibody. The complete sequences and heavy and light chain variable region sequences of DX-2930 are presented below, with signal sequences indicated in italics. CDRs are in bold and underlined.

Amino acid sequence of the heavy chain of DX-2930 (451 amino acids, 49439.02 Da).

NG^SCYFFFVATAFGAASEVOLLESGGGLVOPGGSLRLSCAASGFTFSHYIMMWVROAP

GKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAYRRIGVPR

RDEFDIWGOGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1)

Amino acid sequence of the light chain of DX-2930 (213 amino acids, 23419.08 Da).

MGWSCILFLVATATGAHSOIQMTQSPSTL SASVGDRVTIT CRASQSISSWLAWYQQKPG

KAPKLLIYKA.STLESGVPSRFSGSGSGTEFTLTISSLOPDDFATYYCOQYNTYWTFGOGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)

- 10 045920

Amino acid sequence of the heavy chain variable domain of DX-2930.

EVOLLESGGGLVOPGGSLRLSCAASGFTFSHYIMMWVROAPGKGLEWVSGIYSSGGITV

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGOGTMVTVSS (SEQ ID NO: 3)

Amino acid sequence of the light chain variable domain of DX-2930.

DIOMTOSPSTLSASVGDRVTITCRASQSISSWLAWYOOKPGKAPKLLIYKASTLESGVP

SRFSGSGSGTEFTLTISSLOPDDFATYYCQQYNTYWTFGOGTKVEIK (SEQ ID NO: 4) Table 1

CDRs for DX-2930

CDR Amino acid sequence Heavy chain CDR1 HYIMM (SEQ ID NO: 5) Heavy chain CDR2 GIYSSGGITVYADSVKG (SEQ ID NO: 6) Heavy chain CDR3 RRIGVPRRDEFDI (SEQ ID NO: 7) Light chain CDR1 RASQSISSWLA (SEQ ID NO: 8) Light chain CDR2 KASTLES (SEQ ID NO: 9) Light chain CDR3 QQYNTYWT (SEQ ID NO: 10)

Antibody production

The antibody described in the present invention (eg, DX-2930) can be obtained by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and Greenfield, (2013) Antibodies: A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press.

The sequence encoding an antibody of interest, such as DX-2930, can be stored in a vector in a host cell, and the host cell can then be grown and frozen for later use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to humanize the antibody or to improve affinity (affinity maturation) or other characteristics of the antibody. For example, the constant region can be designed to more closely resemble human constant regions to avoid an immune response if the antibody is used in human clinical trials and treatment. Genetic manipulation of the antibody sequence to achieve greater affinity for the target antigen and greater effectiveness in inhibiting PKa1 activity may be desirable. One skilled in the art will appreciate that it is possible to make one or more polynucleotide changes in an antibody and still retain its specificity for binding to a target antigen.

In other embodiments, fully human antibodies can be produced using commercially available genetically engineered mice to express specific human immunoglobulin proteins. Transgenic animals engineered to achieve a more desirable (e.g., fully human antibody) or more durable immune response can also be used to produce humanized antibodies or human antibodies. Examples of such technology include XenomouseR™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-MouseR™ and TC Mouse™ from Medarex, Inc. (Princeton, N.J.). Alternatively, antibodies can be produced recombinantly using phage display or yeast technology. See, for example, US Pat. Nos. 5,565,332; 5580717; 5733743 and 6265150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire from non-immunized donors.

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

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies and bispecific antibodies, can be produced using, for example, conventional recombinant technology. In one example, DNA encoding monoclonal antibodies specific for a target antigen can be easily isolated or synthesized. The DNA can be placed into one or more expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to produce synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO87/04462. The DNA can then be modified, for example using a coding sequence

- 11 045920 activity for human heavy and light chain constant domains instead of homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently linking to an immunoglobulin coding sequence all or part of the coding sequence of a non-immunoglobulin polypeptide. In this way, it is possible to produce genetically engineered antibodies, such as chimeric or hybrid antibodies; having specificity for binding the target antigen.

Methods developed for the production of chimeric antibodies are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 USA 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

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

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

A single chain antibody can be produced by recombinant technology by combining a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. Preferably, a flexible linker is inserted between the two variable regions. Alternatively, the methods described for the production of single chain antibodies (US Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv library, and PKal-specific scFv clones can be identified from the library using routine procedures. Positive clones can be further screened to identify clones that inhibit PKa1 activity.

Some antibodies, such as Fab, can be produced in bacterial cells, such as E. coli cells (see, for example, Nadkarni, A. et al., 2007 Protein Expr Purif 52 (1):219-29). For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display molecule and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress the stop codon. In this case, Fab does not fuse with gene III protein, but is secreted into the periplasm and/or medium.

Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (eg, scFv) are expressed in a yeast cell, such as Pichia (see, for example, Powers et al., 2001, J. Immunol. Methods. 251:123-35; Schoonooghe S. et al. , 2009 BMC Biotechnol. 9:70; AbdelSalam, 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. One skilled in the art can optimize antibody production in yeast by optimizing, for example, oxygen conditions (see, for example, Baumann K., et al. 2010 BMC Syst. Biol. 4:141), osmolarity (see, for example, Dragosits, M. et al., 2010 BMC Genomics 11:207), temperature (see, for example, Dragosits, M. et al., 2009 J Proteome Res. 8 (3): 1380-92), fermentation conditions (see, e.g. Ning, D. et al. 2005 J. Biochem. and Mol. Biol. 38(3): 294-299), yeast strain (see e.g. Kozyr, A.V. et al. 2004 Mol Biol (Mosk) 38 (6): 1067-75; Horwitz, A.H. et al., 1988 Proc Natl Acad Sci USA 85 (22): 8678-82; Bowdish, K. et al. 1991 J Biol Chem 266 (18): 11901-8 ), overexpression of proteins to enhance antibody production (see, for example, Gasser, B. et al., 2006 Biotechol. Bioeng. 94(2):353-61), the level of acidity of the culture (see, for example, Kobayashi H., et al., 1997 FEMS Microbiol Lett 152 (2):235-42), substrate and/or ion concentrations (see, for example, Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):41-8) . In addition, yeast systems can be used to produce antibodies with extended half-life (see, for example, Smith, BJ. et al. 2001 Bioconjug Chem 12 (5): 750-756).

In one preferred embodiment, the antibodies are produced in mammalian cells. Preferred mammalian host cells for expression of antibody clones or antigen-binding fragments thereof include Chinese hamster cells (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DHFR selectable marker, eg, as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocyte cell lines, eg, NS0 myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol Methods (2004) 289(1-2):65-80) and a transgenic animal cell, e.g.

- 12 045920 gene mammal. For example, the cell is a mammalian epithelial cell.

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

In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, recombinant expression vectors may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector is inserted (see, for example, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector is inserted. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr~ host cells with methotrexate selection/amplification) and the peo gene (for G418 selection).

In an example system for recombinant expression of an antibody or an antigen-binding portion thereof, a recombinant expression vector encoding an antibody heavy chain and an antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as CMV enhancer/AdMLP promoter or SV40 enhancer/AdMLP promoter) to control high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, allowing selection of CHO cells transfected with the vector using methotrexate selection/amplification. The selected transformed host cells are cultured to allow expression of the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to obtain a recombinant expression vector for transfecting host cells, selecting transformants, culturing host cells, and isolating antibody from the culture medium. For example, some antibodies can be isolated by protein A or protein G affinity chromatography.

In the case of antibodies comprising an Fc domain, the antibody production system can produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is a site for modification by antenna type 2 oligosaccharides. This glycosylation has been shown to be required for effector functions mediated by Fcg receptors and complement Clq (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 may also include other eukaryotic post-translational modifications.

Antibodies can also be produced by transgenic animals. For example, US Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed comprising a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes an antibody of interest secreted therein. The antibody can be purified from milk or, for some applications, used directly.

Pharmaceutical compositions

The antibody provided in the present invention (eg, DX-2930) may be present in a composition, for example, a pharmaceutically acceptable composition or a pharmaceutical composition. The antibody provided in the present invention (eg, DX-2930) can be formulated together with a pharmaceutically acceptable carrier. In some embodiments, 150 mg or 300 mg of 30-400 mg DX-2930 antibodies are present in a composition, optionally with a pharmaceutically acceptable carrier, for example, a pharmaceutically acceptable composition or a pharmaceutical composition.

A pharmaceutically acceptable carrier includes any and all of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for subcutaneous, intravenous, intramuscular, parenteral, spinal or epidermal administration (eg, by injection or infusion), although carriers suitable for inhalation and intranasal administration are also provided.

The pharmaceutically acceptable carrier in the pharmaceutical composition described in the present invention may include one or more buffering agents, amino acids and tonicity regulators. Any suitable buffering agent or combination of buffering agents can be used in the pharmaceutical composition described in the present invention to maintain or promote

- 13 045920 maintaining a suitable pH level of the composition. Non-limiting examples of buffering agents include sodium phosphate, potassium phosphate, citric acid, sodium succinate, histidine, Tris and sodium acetate. In some embodiments, buffering agents can be used at a concentration of about 5-100 mM, 5-50 mM, 10-50 mM, 15-50 mM, or about 15-40 mM. For example, one or more buffering agents can be used at a concentration of about 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM , 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM or about 40 mM. In some examples, the pharmaceutically acceptable carrier includes sodium phosphate and citric acid, which can be used at a concentration of about 30 mM and about 19 mM, respectively.

In some embodiments, the pharmaceutically acceptable carrier includes one or more amino acids that can reduce antibody aggregation and/or increase the stability of the antibody during storage prior to administration. Typical amino acids for use in the preparation of the pharmaceutical compositions described in the present invention include, but are not limited to, alanine, arginine, asparagine, aspartic acid, glycine, histidine, lysine, proline or series. In some examples, the concentration of the amino acid in the pharmaceutical composition may be about 5-100 mM, 10-90 mM, 20-80 mM, 30-70 mM, 40-60 mM, or about 45-55 mM. In some examples, the concentration of the amino acid (e.g., histidine) may be about 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM or about 60 mM. In one example, the pharmaceutical composition contains histidine at a concentration of about 50 mM.

Any suitable tonicity regulator can be used to prepare the pharmaceutical compositions described in the present invention. In some embodiments, the tonicity regulator is a salt or an amino acid. Examples of suitable salts include, but are not limited to, sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate and calcium chloride. In some embodiments, the tonicity regulator in the pharmaceutical composition may be present at a concentration of about 10-150 mM, 50-150 mM, 50-100 mM, 75-100 mM, or about 85-95 mM. In some embodiments, the tonicity regulator may be present at a concentration of about 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM , 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM or about 100 mM. In one example, the tonicity regulator may be sodium chloride, which may be present at a concentration of about 90 mM.

The pharmaceutically acceptable carrier in the pharmaceutical compositions provided herein may further include one or more pharmaceutically acceptable excipients. In general, pharmaceutically acceptable excipients are pharmacologically inactive substances. Non-limiting examples of excipients include lactose, glycerin, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin (BSA), dextran, polyvinyl acetate (PVA), hydroxypropyl methylcellulose (HPMC), polyethylenimine (PEI), gelatin, polyvinylpyrrolidone ( PVP), hydroxyethylcellulose (HEC), polyethylene glycol (PEG), ethylene glycol, glycerin, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene sorbitan monooleate (Tween-80), sodium dodecyl sulfate (SDS), polysorbate, polyoxyethylene copolymer, potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, zinc ions, copper ions, calcium ions, manganese ions, magnesium ions, CHAPS, sucrose monolaurate and 2-O-beta- mannoglycerate. In some embodiments, the pharmaceutically acceptable carrier includes an excipient in the range of about 0.001%-0.1%, 0.001%-0.05%, 0.005-0.1%, 0.005%-0.05%, 0.008%-0.05% , 0.008%-0.03% or about 0.009%-0.02%. In some embodiments, the excipient is about 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06 %, 0.07%, 0.08%, 0.09% or about 0.1%. In some embodiments, the excipient is polyoxyethylene sorbitan monooleate (Tween-80). In one example, the pharmaceutically acceptable carrier contains 0.01% Tween-80.

In some examples, the pharmaceutical composition described in the present invention includes an anti-pKal antibody, as also described in the present invention (for example, DX-2930), and one or more of sodium phosphate (for example, dibasic sodium phosphate dihydrate), citric acid ( for example, citric acid monohydrate), histidine (for example, L-histidine), sodium chloride, and Polysorbate 80. For example, the pharmaceutical composition may include an antibody, sodium phosphate, citric acid, histidine, sodium chloride, and Polysorbate 80. In some examples, the antibody is formulated in about 30 mM sodium phosphate, about 19 mM citric acid, about 50 mM histidine, about 90 mM sodium chloride and about 0.01% Polysorbate 80. The concentration of antibody (for example, DX-2930) in the composition may be about 150 mg/ml or 300 mg/ml. In one example, the composition comprises or consists of about 150 mg DX-2930 per ml solution, about 30 mM dibasic sodium phosphate dihydrate, about 19 mM (e.g., 19.6 mM) citric acid monohydrate, about 50 mM L-histidine, about 90 mM sodium chloride and about 0.01% Polysorbate 80. In another example, the composition includes or consists of about 300 mg DX-2930 per ml solution, about 30 mM dibasic sodium phosphate dihydrate, about 19 mM (e.g., 19.6 mM ) citric acid monohydrate, about 50 mM L-histidine, about

- 14045920 mM sodium chloride and about 0.01% Polysorbate 80.

A pharmaceutically acceptable salt is a salt that retains the desired biological activity of the compound and does not have any undesirable toxic effects (see, for example, Berge, S. M., et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, orthophosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, etc., as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Basic addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines such as N.N'-dibenzylethylenediamine. Nmethylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

Compositions can take many forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg, injectable and infusable solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form may depend on the intended route of administration and therapeutic use. Many compositions are in the form of injectable or infused solutions, such as compositions similar to those used for administration to humans with antibodies. An example of a route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, plasma kallikrein binding protein is administered via intravenous infusion or injection. In another preferred embodiment, plasma kallikrein binding protein is administered by intramuscular or subcutaneous injection. In another preferred embodiment, plasma kallikrein binding protein is administered via intraperitoneal injection.

The phrases parenteral and parenterally administered as used herein mean methods of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intratracheal, intracapsular, intraorbital, intracardiac, intradermal , intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In some embodiments, the antibody is administered subcutaneously.

The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in a suitable solvent with one or a combination of the ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, whereby a powder of the active ingredient with any additional desired ingredient is obtained from a previously sterilized solution by filtration. Suitable fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition an absorption delaying agent, for example, monostearate salts and gelatin.

The antibody provided herein (eg, DX-2930) can be administered in a variety of ways, including intravenous injection or infusion. For example, for some therapeutic applications, the antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5 or 1 mg/min to achieve a dose of about 1 to 100 mg/m ² or 7 to 25 mg/m ² . The route and/or method of administration may vary depending on the desired results. In some embodiments, the active compound can be prepared using a carrier that will protect the compound from rapid release, for example, in the form of a controlled release composition, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. There are many methods available for preparing such compositions. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., 1978, Marcel Dekker, Inc., New York.

Pharmaceutical compositions can be administered using medical devices. For example, in one embodiment, a pharmaceutical composition provided herein can be administered using a device, such as a needle-free subcutaneous injection device, pump, or implant.

- 15 045920

In some embodiments, an antibody provided herein (eg, DX-2930) can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed in the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, US Pat. Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes may include one or more moieties that are selectively transported to specific cells or organs, thereby enhancing delivery of the target drug (see, e.g., V. V. Ranade, 1989, J. Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the therapeutic situation. It is particularly preferred that parenteral compositions are formulated in unit dosage form for ease of administration and uniform dosage. In the context of the present invention, unit dosage form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to achieve the desired therapeutic effect, together with the desired pharmaceutical carrier. Requirements for unit dosage forms may be determined and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect sought to be achieved, and (b) the limitations inherent in the field of compounding such active compound for the treatment of sensitivity in individuals.

An exemplary non-limiting range for a therapeutically or prophylactically effective amount of an antibody described in the present invention (eg, DX-2930) is about 150 mg or 300 mg. As one skilled in the art will appreciate, the therapeutically or prophylactically effective amount of antibody may be lower for a pediatric subject than for an adult subject. In some embodiments, the effective amount that is administered to a pediatric subject is a fixed dose or a weight-based dose. In some embodiments, an effective amount that is less than about 150 mg or 300 mg is administered to a pediatric subject. In some embodiments, a therapeutically or prophylactically effective amount of the antibody is administered every two weeks or every four weeks during the first treatment period. In some embodiments, the antibody may be administered to the subject during a second treatment period. In some embodiments, the therapeutically or prophylactically effective amount of antibody in the first treatment period is different from the therapeutically or prophylactically effective amount of antibody in the second treatment period. In some embodiments, the therapeutically or prophylactically effective amount of antibody in the first treatment period is 150 mg, and the therapeutically or prophylactically effective amount of antibody in the second treatment period is 300 mg. In some embodiments, the therapeutically or prophylactically effective amount of antibody in the first treatment period is the same as the therapeutically or prophylactically effective amount of antibody in the second treatment period. In one example, the therapeutically or prophylactically effective amount of antibody in the first treatment period and the second treatment period is 300 mg.

In some embodiments, a typical, non-limiting range for a therapeutically or prophylactically effective amount of an antibody described herein (eg, DX-2930) is about 300 mg. In some embodiments, a therapeutically or prophylactically effective amount of the antibody is administered in a single dose. If a subject experiences an exacerbation of HAE, the antibody may be further administered to the subject in multiple doses, such as about 300 mg administered every two weeks.

Sets

An antibody described in the present invention (eg, DX-2930) may be provided in a kit, for example, as a kit component. For example, the kit includes (a) a DX-2930 antibody, for example, a composition (eg, a pharmaceutical composition) comprising the antibody, and optionally (b) information material. The information material may be descriptive, instructional, marketing or other material related to the method of the present invention and/or the use of an antibody as described in the present invention (eg, DX-2930), for example, for the method of the present invention . In some embodiments, the kit contains one or more doses of DX-2930. In some embodiments, one or more doses are 150 mg or 300 mg.

The information material from the set is not limited in its form. In one embodiment, the information material may include information about the preparation of the compound, molecular weight of the compound, concentration, expiration date, lot or place of receipt, etc. In one embodiment, the information material relates to the use of an antibody for the treatment, prevention or diagnosis of disorders and conditions, for example, a disease or condition associated

- 16 045920 bath with plasma kallikrein.

In one embodiment, the information material may include instructions for administering an antibody described herein (e.g., DX-2930) in a manner suitable for carrying out the methods provided herein, e.g., at a suitable dose, dosage form, using a suitable method, or administration schedule (eg, using the dose, dosage form, schedule or route of administration described in the present invention). In another embodiment, the information material may include instructions for administering an antibody described in the present invention (eg, DX-2930) to a suitable subject, for example, a person, for example, a person suffering from or at risk of developing a disease or condition associated with plasma kallikrein. For example, the material may include instructions for administering an antibody described herein (e.g., DX-2930) to a patient with a disorder or condition described herein, e.g., plasma kallikrein-associated disease, e.g., in accordance with an administration schedule, presented in the present invention. Information material from the sets is not limited in its form. In many cases, information material, such as instructions, is presented in printed form, but may also be presented in other formats, such as machine-readable material.

The antibody described in the present invention (eg, DX-2930) can be presented in any form, for example, liquid, dried or lyophilized form. Preferably, the antibody is substantially pure and/or sterile. If the antibody is presented in a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. If the antibody is in dried form, reconstitution is typically accomplished by adding a suitable solvent. A solvent, such as sterile water or a buffer, may optionally be provided in the kit.

The kit may include one or more containers for a composition containing an antibody described in the present invention (eg, DX-2930). In some embodiments, the kit contains separate containers, dividers or compartments for composition and information material. For example, the composition may be contained in a bottle, container or syringe, and the information material may be contained with the container. In other embodiments, the individual elements of the set are contained in a single, non-divided container. For example, the composition is contained in a bottle, container or syringe with attached information material in the form of a label. In some embodiments, the kit includes a plurality (eg, a kit) of individual containers, each of which contains one or more unit dosage forms (eg, a dosage form provided herein) of an antibody described herein (eg, DX-2930). For example, a kit includes a plurality of syringes, ampoules, foil pouches or blisters, each containing one unit dose of an antibody of the present invention (eg, DX-2930). Kit containers may be sealed, waterproof (eg, impervious to changes in humidity or evaporation), and/or lightproof.

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 that dispenses metered doses of the antibody. The present disclosure also relates to a method for preparing a kit, for example, by combining the components described in the present invention.

Treatment

In some aspects, the disclosure provides use of an antibody described in the present invention (eg, DX-2930) in the treatment of HAE.

(i) Hereditary angioedema.

Hereditary angioedema (HAE) is also known as angioedema, C1-esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioedema (HANE). HAE is characterized by unpredictable, recurring attacks of severe subcutaneous or submucosal swelling (angioedema), which can affect, for example, the limbs, face, genitals, gastrointestinal tract and respiratory tract (Zuraw, 2008). Symptoms of HAE include, for example, swelling of the hands, feet, lips, eyes, tongue and/or throat; airway blockage, which may include swelling of the throat, sudden hoarseness, and/or cause death by suffocation (Bork et al., 2012; Bork et al., 2000). Approximately 50% of all patients with HAE will experience a laryngeal attack during their lifetime, and there is no way to predict which patients are at risk for a laryngeal attack (Bork et al., 2003; Bork et al., 2006). Symptoms of HAE also include repeated episodes of abdominal cramps without an obvious cause; and/or intestinal swelling, which may be severe and may result in abdominal cramping, vomiting, dehydration, diarrhea, pain, shock and/or intestinal symptoms similar to abdominal emergencies, which may lead to unnecessary surgery (Zuraw, 2008 ). The swelling may last up to five days or more. About a third of people with HAE develop a non-pruritic rash called rheumatitis during an attack.

- 17 045920 erythema. Most patients experience several attacks per year.

HAE is an orphan disorder whose exact distribution is unknown, but current estimates range from 1 in 10,000 to 1 in 150,000 people, with many authors agreeing that 1 in 50,000 is probably the closest estimate (Bygum, 2009; Goring et al ., 1998; Lei et al., 2011; Nordenfelt et al., 2014; Roche et al., 2005).

Plasma kallikrein plays a critical role in the pathogenesis of HAE flares (Davis, 2006; Kaplan and Joseph, 2010). In normal physiology, C1-INH regulates the activity of plasma kallikrein as well as numerous other proteases such as C1r, C1s, factor XIa and factor XIIa. Plasma kallikrein regulates the release of bradykinin from high molecular weight kininogen (HMWK). Due to C1-INH deficiency in HAE, uncontrolled plasma kallikrein activity occurs and leads to excessive generation of bradykinin. Bradykinin is a vasodilator thought to be responsible for the characteristic symptoms of HAE, such as localized swelling, inflammation, and pain (Craig et al., 2012; Zuraw et al., 2013).

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

Trauma or stress, such as dental procedures, illness (such as viral illnesses such as colds and flu), menstruation, and surgery can trigger an episode of angioedema. To prevent acute attacks of HAE, patients may try to avoid specific stimuli that have previously triggered attacks. However, in many cases the attack occurs in the absence of a known trigger. Typically, HAE symptoms first appear in childhood and worsen during puberty. On average, untreated individuals have attacks every 1 to 2 weeks, and most episodes last for approximately 3 to 4 days (ghr.nlm.nih.gov/condition/hereditary-angioedema). The frequency and duration of attacks vary considerably among people with hereditary angioedema, even among people in the same family.

There are three types of HAE, known as types I, II and III, all of which can be treated with the methods described in the present invention. HAE is thought to affect 1 in 50,000 people, with type I accounting for approximately 85 percent of cases, type II accounting for approximately 15 percent of cases, and type III being very rare. Type III is the most recently described form and was initially thought to occur only in women, but families with affected men have been identified.

HAE is inherited in an autosomal dominant manner such that an affected individual may inherit the mutation from one affected parent. New mutations in the gene can also occur, and thus HAE can also develop in people without a family history of the disorder. It is believed that 20-25% of cases are the result of a new spontaneous mutation.

Mutations in the SERPING1 gene cause hereditary angioedema type I and type II. The SERPING1 gene provides instructions for the production of the inhibitory protein C1, important for controlling inflammation. A C1 inhibitor blocks the activity of certain proteins that stimulate inflammation. Mutations that cause hereditary angioedema type I result in decreased levels of the C1 inhibitor in the blood. Conversely, mutations causing type II result in the production of a C1 inhibitor that functions abnormally. Approximately 85% of patients have type I HAE, characterized by very low production of functionally normal C1-INH protein, while the remaining approximately 15% of patients have type II HAE and have normal or elevated levels of functionally impaired C1-INH (Zuraw, 2008). Without the correct levels of functional C1 inhibitor, excessive amounts of bradykinin are produced from high molecular weight kininogen (HMWK), and there is increased vascular leakage mediated by bradykinin binding to the B2 receptor (B2-R) on the surface of endothelial cells (Zuraw, 2008). Bradykinin promotes inflammation by increasing the leakage of fluid through the walls of blood vessels into body tissue. Excessive accumulation of fluids in body tissues causes episodes of swelling seen in people with hereditary angioedema type I and type II.

Mutations in the F12 gene are associated with some cases of hereditary angioedema type III. The F12 gene provides instructions for the production of coagulation factor XII. In addition to its critical role in blood clotting (coagulation), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Specific mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is produced and the blood vessel walls become more permeable, leading to episodes of swelling. The cause of other cases of hereditary angioedema type III remains unknown. Mutations in one or more as yet unidentified genes may be responsible for the disorder in these cases.

HAE may be similar to other forms of angioedema resulting from allergies or other medical conditions, but differs significantly from them in terms of cause and treatment. When hereditary angioedema is misdiagnosed as an allergy, it is most often treated with antihistamines, steroids and/or epinephrine, which are generally ineffective for HAE, although epinephrine can be used in life-threatening reactions. Incorrect diagnosis also leads to unnecessary diagnostic surgery

- 18 045920 intervention in patients with abdominal edema, and in some patients with HAE, abdominal pain is misdiagnosed as psychosomatic.

Like adults, children with HAE may suffer from repeated and debilitating attacks. Symptoms can appear very early in childhood, and upper airway angioedema has been reported in patients with HAE as young as 3 years (Bork et al., 2003). In one study of 49 patients with pediatric HAE, 23 had at least one episode of airway angioedema before the age of 18 years (Farkas, 2010). There is a huge unmet need for medical treatment among children with HAE, especially adolescents, as the disease typically worsens after puberty (Bennett and Craig, 2015; Zuraw, 2008).

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

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

A purified C1 inhibitor obtained from human blood has been used in Europe since 1979. Several C1 inhibitor drugs are now available in the US, and two C1 inhibitor products are now available in Canada. Behring R (CSL Behring), which is pasteurized, is approved by the F.D.A. in 2009 for acute attacks. Nanofiltered Cinriz (ViroPharma) is F.D.A. approved. in 2008 for prevention. Rucin (Pharming) is a recombinant C1 inhibitor under development that does not carry the risk of infectious disease transmission from blood-borne human pathogens.

Treatment for an acute attack of HAE may also include medications for pain relief and/or IV (intravenous) fluids.

Other treatments may stimulate C1 inhibitor synthesis or reduce C1 inhibitor uptake. Androgenic drugs such as danazol may reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

Helicobacter pylori can trigger abdominal attacks. Antibiotics to treat H. pylori will reduce abdominal attacks.

Newer drugs target the contact cascade. Ecallantide (KALBITOR®, DX-88, Dyax) inhibits plasma kallikrein and is approved in the United States. Icatibant (FIRAZYR®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

The diagnosis of HAE may be based, for example, on family history and/or blood tests. Laboratory findings associated with HAE types I, II and III are described, for example, in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925. In HAE type I, the level of C1 inhibitor is reduced, as is the level of C4, while the level of C1q is normal. In HAE type II, C1 inhibitor levels are normal or elevated; however, the function of the C1 inhibitor is abnormal. C4 levels are decreased and C1q levels are normal. In type III, C1 inhibitor, C4, and C1q levels may all be normal.

Symptoms of HAE can be assessed, for example, using questionnaires, such as questionnaires completed by patients, clinicians or family members. Such questionnaires are known in the art and include, for example, visual analogue scales. See, for example, McMillan, C.V. et al. Patient. 2012;5(2):113-26. In some embodiments, the subject has HAE type I or HAE type II. HAE type I or HAE type II can be diagnosed using any method known in the art, such as a clinical history suggestive of HAE (eg, episodes of subcutaneous or mucosal nonpruritic swelling) or diagnostic testing (eg, C1-INH functional testing and evaluation level C4) (ii) Treatment of HAE with anti-pKal antibodies The present invention relates to methods of treating (eg, improving, stabilizing or eliminating one or more symptoms) hereditary angioedema (HAE) by administering an antibody described in the present invention (eg, therapeutically effective amount of the antibody described in the present invention) to a subject suffering from or suspected of having HAE, for example, in accordance with the dosage regimen described in the present invention. In addition, the present invention provides methods for treating HAE by administering an antibody described herein (e.g., a therapeutically effective amount of an antibody described herein), for example, in accordance with the dosage regimen described in the present invention, or in combination with a second therapy, for example, using one other agent, for example, described in the present invention. The present invention also provides methods for preventing HAE or a symptom thereof by administering an antibody described herein (e.g., a prophylactically effective amount of an antibody described herein) to a subject at risk for developing HAE (e.g., a subject who has a family member with HAE or with a genetic predisposition to it), for example, in accordance with the dosage regimen described in the present invention. In some examples, the subject may be a human patient without

- 19 045920 symptoms of HAE at the time of treatment. In some embodiments, the subject is a human patient with HAE type I or HAE type II. In some embodiments, the subject is a human patient who has experienced at least two (eg, 2, 3, 4, 5 or more) attacks of HAE in the year prior to treatment.

Treatment involves administering an amount effective to alleviate, ameliorate, treat, ameliorate, or influence the disorder, symptoms of the disorder, or susceptibility to the disorder. Treatment can also slow the onset of a disease, such as preventing the onset of a disease or preventing a disease or condition from getting worse.

Methods of administering DX-2930 antibodies are also described in the Pharmaceutical Compositions section. The appropriate dose of antibody used may depend on the age and body weight of the subject and the particular drug used. The antibody can be used as a competitive agent to inhibit, reduce an undesirable interaction, for example, between plasma kallikrein and its substrate (eg, factor XII or HMWK). The dose of the antibody may be an amount sufficient to block 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% of plasma kallikrein activity in a patient. especially at the site of the disease. In some embodiments, 150 mg or 300 mg of antibody is administered every two weeks or every four weeks. In some embodiments, 300 mg of antibody is administered in a single dose. If a subject experiences an attack of HAE after a single dose, the antibody can be administered at 300 mg every two weeks.

In one embodiment, the antibodies are used to inhibit plasma kallikrein activity (eg, inhibit at least one plasma kallikrein activity, eg, decrease the production of factor XIIa and/or bradykinin), for example, in vivo. Binding proteins can be used as such or conjugated to an agent, for example, a cytotoxic drug, cytotoxin, enzyme or radioactive isotope.

Antibodies can be used directly in vivo to eliminate antigen-expressing cells through natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). Antibodies provided herein may include a complement-fixing effector domain, such as Fc portions of IgG1, -2, or -3, or corresponding complement-fixing portions of IgM. In one embodiment, a population of target cells is treated ex vivo with an antibody provided herein and suitable effector cells. Treatment can be supplemented by adding complement or serum containing complement. In addition, phagocytosis of target cells coated with an antibody provided herein can be improved by binding complement proteins. In another embodiment, target cells coated with an antibody comprising a complement-binding effector domain are lysed with complement.

Methods of administering DX-2930 antibodies are described in the Pharmaceutical Compositions section. Suitable dosages of the molecules used will depend on the age and body weight of the subject and the particular drug used. Antibodies can be used as competitive agents to inhibit or reduce an undesirable interaction, for example, between a natural or pathological substance and plasma kallikrein.

A therapeutically effective amount of an antibody described in the present invention can be administered to a subject having, suspected of, or at risk of developing HAE, thereby providing treatment (e.g., alleviating, ameliorating a symptom or sign of the disorder, slowing, stabilizing, and/or delaying the progression of diseases) disorders.

The antibody described in the present invention can be administered in a therapeutically effective amount. A therapeutically effective amount of an antibody is an amount effective after administration of a single or more doses to a subject in treating the subject, eg, curing, alleviating, or improving at least one symptom of a disorder in the subject to an extent that is greater than would be expected in the absence of such treatment.

Dosage regimens may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased depending on the therapeutic situation. In other examples, a bolus may be administered followed by multiple doses over a period of time, or the dose may be proportionally decreased or increased as the therapeutic situation dictates. In other examples, the dose may be divided into multiple doses and administered over a period of time. It is particularly preferred that parenteral compositions are formulated in unit dosage form for ease of administration and uniform dosage. In the context of the present invention, unit dosage form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to achieve the desired therapeutic effect, together with the desired pharmaceutical carrier.

In some embodiments, an antibody described in the present invention is administered

- 20 045920 dosing pressure during the first treatment period. In some embodiments, the antibody is administered in the first treatment period in multiple doses. During this period, a therapeutically or prophylactically effective amount of antibody (eg, DX-2930) may be about 150 mg or 300 mg and administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks or longer. In some embodiments, a therapeutically or prophylactically effective amount of antibody (eg, DX-2930) may be about 150 mg or 300 mg and administered every two weeks or every four weeks. In some embodiments, a therapeutically or prophylactically effective amount is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times or more. In some embodiments, the first treatment period is 26 weeks. In some embodiments, the therapeutically or prophylactically effective amount is 150 mg and is administered to the subject every four weeks (eg, every four weeks for 26 weeks, resulting in a total of 7 doses delivered). In some embodiments, the therapeutically or prophylactically effective amount is 300 mg and is administered to the subject every two weeks (eg, every two weeks for 26 weeks, resulting in a total of 13 doses delivered).

In one example, the first treatment period is 26 weeks, and the antibody is administered on day 0, day 28, day 56, day 84, day 112, day 140, and day 168. In another example, the first treatment period is 26 weeks, and the antibody is administered on day 0, day 14, day 28, day 42, day 56, day 70, day 84, day 98, day 112, day 126, day 140, day 154 and day 168. It will be understood by those skilled in the art that the following treatment regimen allows a ±4 day window (for example, ±3 days, ±2 days, or ±1 day). For example, a dose administered on days 10-18 would be covered by the day 14 dose noted above.

In some embodiments, a therapeutically or prophylactically effective amount is administered in a dosage regimen during a second treatment period after the first treatment period. In some embodiments, the therapeutically or prophylactically effective amount is different in the first treatment period and the second treatment period. In some embodiments, the therapeutically or prophylactically effective amount for the second treatment period is about 300 mg. During this period, the antibody can be administered in multiple doses of about 300 mg, such as 300 mg administered every two weeks. In some embodiments, in the second treatment period, multiple doses of the antibody are administered at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times. In some embodiments, the second treatment period is 26 weeks. In some embodiments, the antibody is administered at a dose of about 300 mg every two weeks for 26 weeks (eg, resulting in 13 doses delivered). In some embodiments, a single first dose of the second treatment period is administered approximately two weeks after the last dose of the first treatment period.

In any of the embodiments described in the present invention, the time of administration of the antibody is approximate and may include three days before and three days after the specified day (for example, administration every two weeks covers administration on day 11, day 12, day 13, day 14 , day 15, day 16 or day 17).

In some embodiments, an antibody described in the present invention is administered in a single dose of about 300 mg to a subject who has undergone prior HAE treatment (first treatment), such as multi-dose treatment with the same anti-pKal antibody as described in the present invention (e.g., DX -2930). If a subject experiences an exacerbation of HAE after a single dose, the subject may receive antibody treatment in multiple doses of about 300 mg every two weeks for a suitable period, for example, 26 weeks. In some embodiments, the first of multiple doses is administered no later than one week after the HAE attack (eg, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the HAE attack). In some embodiments, the antibody is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times or more.

Previous treatment for HAE may include the same antibody as described in the present invention (eg, DX-2930). In some embodiments, prior HAE treatment may include multiple doses of DX-2930 every two weeks or every four weeks. In some embodiments, DX-2930 is administered to a subject (eg, subcutaneously) at a dose of 150 mg every two weeks, at a dose of 300 mg every two weeks, or at a dose of 300 mg every four weeks. In one example, the subject was previously administered the antibody every two weeks or four weeks

- 21 045920 for 26 weeks before the administration of a single dose of the antibody. In some embodiments, multiple doses of the pretreatment antibody are administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times. In some embodiments, the antibody was previously administered on day 0, day 28, day 56, day 84, day 112, day 140, and day 168. In some embodiments, a single dose of about 300 mg of antibody is administered approximately two weeks after the last dose of the prior treatment . In one example, a single dose of the second treatment period is administered on day 182 of the first treatment period.

In any of the embodiments described in the present invention, the time of administration of the antibody is approximate and may include three days before and three days after the specified day (for example, administration every two weeks covers administration on day 11, day 12, day 13, day 14 , day 15, day 16 or day 17).

In some embodiments, prior to administration of the antibody in accordance with any of the methods described in the present invention, the subject may be assessed to establish a baseline frequency of HAE attacks. This evaluation period can be called an introductory period. In some embodiments, the baseline frequency of HAE attacks must meet or exceed a minimum number of HAE attacks over a given period of time. In one example, the subject experiences at least one attack of HAE during the four-week run-in period prior to the first antibody administration. In another example, the subject experiences at least two attacks of HAE during the eight-week run-in period prior to the first antibody administration.

Any of the subjects provided in the present invention may undergo pre-treatment with HAE, such as prophylactic or therapeutic treatment for HAE. Aspects of the present disclosure also provide methods of administering an antibody described herein (eg, DX-2930) to a subject who has received one or more prior HAE treatments. In some embodiments, the prior HAE treatment is a treatment that includes an antibody described in the present invention (eg, DX-2930). In some embodiments, the subject has previously been administered multiple doses of DX-2930 every two weeks or every four weeks. In some embodiments, the subject has previously been administered 150 mg of DX-2930 every two weeks. In some embodiments, the subject has previously been administered 300 mg of DX-2930 every two weeks. In some embodiments, the subject has previously been administered 300 mg of DX2930 every four weeks. In some embodiments, multiple doses of the pretreatment antibody are administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times.

In some embodiments, the subject has received one or more prior treatments for HAE, which may include any therapeutic agent for treating HAE known in the art. Typical anti-HAE agents include, but are not limited to, C1 inhibitors (eg, Cinriz®, Berinert® or Ruconest®), plasma kallikrein inhibitors (eg, Kalbitor®), bradykinin receptor inhibitors (eg, Firazyr®), attenuated androgens (eg danazol), antifibrinolytic agent (eg tranexamic acid). In some examples, a subject may undergo a period of tapering before receiving treatment with an anti-pKal antibody described in the present invention. A taper period refers to the period, prior to anti-pKal antibody treatment, during which a subject who is undergoing anti-HAE treatment (eg, C1-INH, oral androgen, and/or oral antifibrinolytic agents) is gradually reduced in dosage, frequency, or both anti-HAE agents so that the subject can gradually transition from prior HAE treatment to treatment with the anti-pKal antibody described in the present invention. In some embodiments, tapering involves a gradual or stepwise method of reducing the dosage and/or frequency of administration of a prior treatment. The dosage reduction period may last 2-4 weeks and may vary depending on individual patient factors. In some examples, prior treatment is stopped before anti-pKal antibody treatment is initiated. In other examples, the prior treatment may end within a suitable time interval (eg, 2 weeks, 3 weeks, or 4 weeks) after the subject receives its first dose of the anti-pKal antibody.

Alternatively, a subject receiving prior HAE treatment may be switched to treatment with an anti-pKαl antibody described herein immediately without a dosage reduction period.

In other embodiments, the subject does not have any prior HAE treatment prior to the first treatment, first treatment period, and/or subsequent single and multiple doses as described herein (second treatment period). In some embodiments, implementation

- 22 045920 leniya the subject does not receive any treatment other than treatment with the antibody described in the present invention during the first treatment period and/or during the second treatment period. In some embodiments, the subject is free of any prior HAE treatment for at least two weeks (e.g., at least two, three, four, five weeks or more) before the first treatment or first treatment period, during the first treatment, or the first treatment period and/or during the second treatment period. In some embodiments, the subject has not had long-term HAE prophylaxis (eg, C1 inhibitor, attenuated androgens, antifibrinolytics) for at least two weeks prior to the first treatment or first treatment period, during the first treatment period, and/or during the second period treatment. In some embodiments, the subject does not take an HAE treatment including an angiotensin-converting enzyme (ACE) inhibitor for at least four weeks prior to the first treatment or first treatment period, during the first treatment period, and/or during the second treatment period. In some embodiments, the subject does not take the estrogen-containing drug for at least four weeks before the first treatment or first treatment period, during the first treatment period, and/or during the second treatment period. In some embodiments, the subject is not taking androgens (eg, stanozolol, danazol, oxandrolone, methyltestosterone, testosterone) for at least two weeks prior to the first treatment or first treatment period, during the first treatment period, and/or during the second treatment period.

Any of the methods described in the present invention may further include monitoring the patient for side effects (eg, increased levels of creatine phosphatase) and/or levels of pKal inhibition by the antibody (eg, serum or plasma antibody concentration or pKal activity level) before and after treatment or during the course of treatment. If one or more side effects are observed, the dose of the antibody may be reduced or treatment may be discontinued. If the level of inhibition is below the minimum therapeutic level, additional doses of the antibody can be administered to the patient. Patients may also be assessed for antibody generation against the administered antibody; C1 inhibitor, C4 and/or C1q activity; the quality of life; frequency of any HAE attacks, health-related quality of life, anxiety and/or depression (eg, Hospital Anxiety and Depression Scale (HADS)), work productivity (eg, Work Disability and Activities of Daily Living (WPAI) questionnaire), preference for subcutaneous administration antibodies (eg, D-2930) compared with other injections, quality of life (eg, angioedema-quality of life (AE-QOL), report with 5 EuroQoL Group measurement results).

In some embodiments, plasma or serum concentrations of an antibody (eg, DX-2930) may be measured during a course of treatment (eg, after an initial dose) to assess the effectiveness of treatment. If the antibody concentration in plasma or serum is less than about 80 nM, a subsequent dose may be required, which may be the same as or greater than the initial dose. The concentration of an antibody in plasma or serum can be measured by determining the level of antibody protein in a sample of plasma or serum obtained from a subject, for example, using an immunoassay or MS analysis. The concentration of antibody in plasma or serum can also be measured by determining the inhibitory pKal level in a sample of plasma or serum obtained from a subject being treated with the antibody. Such assays may include a synthetic substrate assay or a Western blot assay to measure cleaved kininogen, as described in the present invention.

Alternatively or in addition, plasma or serum creatine kinase levels and/or one or more coagulation parameters (eg, activated partial thromboplastin time (aPTT), prothrombin time (PT), bleeding events) can be monitored during the course of treatment. If plasma or serum creatine kinase levels are found to increase during treatment, the antibody dose may be reduced or treatment may be discontinued. Likewise, if one or more coagulation parameters are found to change significantly during treatment, the dosage of the antibody may be adjusted or treatment may be discontinued.

In some embodiments, the optimal dose (eg, optimal prophylactic dose or optimal therapeutic dose) of an antibody (eg, DX-2930) can be determined as follows. The antibody is administered to the subject in need of treatment at an initial dose. The antibody concentration in the subject's plasma is measured. If the plasma concentration is lower than 80 nM, the dose of the antibody is increased with subsequent administration. A dose of antibody that maintains plasma antibody concentrations greater than about 80 nM may be selected as the optimal dose for the subject. A subject's creatine phosphokinase level may be monitored during the course of treatment, and the optimal dose for that subject may be further adjusted based on the creatine phosphokinase level, for example, the antibody dose may be reduced if an increase in creatine phosphokinase is observed during treatment.

(iii) Combination therapies.

An antibody provided herein (eg, DX-2930) may be administered in combination with one or more other therapeutic agents to treat a disease or condition associated with plasma kallikrein activity, such as the disease or condition described herein. For example, the antibody described in the present invention (for example, DX

- 23 045920

2930), can be used therapeutically or prophylactically (e.g., before, during, or after a course of treatment) with another Fab or IgG against plasma kallikrein (e.g., another Fab or IgG described in the present invention), another plasma kallikrein inhibitor, peptide inhibitor, small molecule inhibitor or surgery. Examples of plasma kallikrein inhibitors that can be used in combination therapy with plasma kallikrein binding antibodies described in the present invention include plasma kallikrein inhibitors described, for example, in WO 95/21601 or WO 2003/103475.

One or more plasma kallikrein inhibitors can be used in combination with an antibody of the present invention (eg, DX-2930). For example, the combination may result in a reduction in the required dose of the inhibitor, thereby reducing side effects.

An antibody provided herein (eg, DX-2930) can be administered in combination with one or more currently known therapeutic agents for the treatment of HAE. For example, antibody DX-2930 may be used in conjunction with a second anti-HAE therapeutic such as ecallantide, a C1-esterase inhibitor (eg, CINRIZ™), aprotinin (TRASILOL®), and/or a bradykinin B2 receptor inhibitor (eg, icatibant (FIRAZIR®) )).

The term combination refers to the use of two or more agents or therapies to treat the same patient, where the use or effect of the agents or therapies overlap in time. The agents or therapies may be administered simultaneously (eg, as a single composition administered to a patient or as two separate compositions administered simultaneously) or sequentially in any order. Sequential administrations are administrations given at different times. The time between administration of one agent and another may be minutes, hours, days or weeks. The plasma kallikrein binding antibody provided herein can also be used to reduce the dose of another therapeutic agent, for example, to reduce side effects associated with another agent administered. Thus, the combination may include administering the second agent at a dose at least 10, 20, 30 or 50% lower than that which would be used in the absence of the plasma kallikrein binding antibody. In some embodiments, a subject may receive a C1 inhibitor as an IV loading dose or SC dose concurrently with a first dose of an anti-pKal antibody (eg, DX-2930) as described herein. The subject may then continue treatment with the anti-pKal antibody (without additional doses of C1 inhibitor).

Combination therapy may involve administering an agent that reduces the side effects of other therapies. The agent may be an agent that reduces side effects in the treatment of plasma kallikrein-related disease, (iv) Assays to evaluate the treatment regimen

Also provided within the scope of this disclosure are assay methods for assessing the effectiveness of any of the treatments described herein. In some embodiments, plasma or serum concentrations of one or more biomarkers (eg, 2-chain HMWK) associated with HAE can be measured before and/or during a course of treatment (eg, after an initial dose) to assess the effectiveness of treatment. In some embodiments, the plasma or serum concentration (level) of one or more HAE-associated biomarkers obtained at a post-dose time point is compared with the biomarker concentration in a sample obtained at an earlier post-dose time point or before the initial dose. . In some embodiments, the biomarker is 2-HMWK.

The level of a biomarker can be measured by detecting the biomarker in a plasma or serum sample obtained from a subject, for example, using an immunoassay such as a Western blot or ELISA using an antibody that specifically detects the biomarker. In some embodiments, the level of 2-HWMK in a plasma or serum sample obtained from a subject is assessed using an immunoassay. Antibodies for use in immunoassays for the detection of 2-HWMK are known in the art, and the selection of such an antibody for use in the methods described herein will be apparent to one skilled in the art.

Without further elaboration, it is believed that one skilled in the art, based on the above description, will be able to use the present invention to its fullest extent. Accordingly, the following specific embodiments should be construed as illustrative only and not as limiting of the remainder of the disclosure in any way. All publications cited in the present invention are incorporated by reference for the purposes or subject matter described in the present invention.

Examples

Example 1: Double-blind study to evaluate the efficacy and safety of DX-2930 using multiple doses.

General description of the study.

A phase 3, multicenter, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of DX-2930 in preventing acute attacks in patients with type I and type II HAE is described. This double-blind study may be followed by

- 24 045920 for the next period of treatment. Typically included are subjects 12 years of age or older with a documented diagnosis of HAE type I or type II who experience at least 1 attack within 4 weeks during the run-in period.

Long-term preventive treatment (LTP).

Washing out.

After informed consent, subjects undergo screening assessments. Screened subjects who are undergoing long-term HAE prophylactic therapy must undergo a minimum 2-week washout period before commencing the run-in period. LTP washout is permitted provided that the investigator determines that it would not result in an unreasonable safety risk to the subject and the subject is at least 18 years of age. These criteria ensure that patients who should remain on LTP are not excluded from this study, but allow the inclusion of eligible patients with severe illness, minimizing their time to completion of LTP. Current treatment guidelines recognize two different standards of treatment approaches for HAE, which include LTP and on-demand therapy (Cicardi et al., 2012; Craig et al., 2012; Zuraw et al., 2013). Subjects are allowed to treat acute attacks of HAE throughout the study. Thus, those subjects who stop LTP to enroll in the study and who are subsequently randomized to placebo continue to be monitored without a reduction in the standard of care. Confirmation that the subject has successfully completed the 2-week washout period is required before they can enter the run-in period.

Introductory period.

Screened subjects who are either not on long-term preventive therapy for HAE or have completed the required washout period undergo a run-in period of 4 weeks to determine the baseline frequency of HAE attacks. Only subjects meeting the minimum baseline frequency with at least 1 confirmed attack of HAE in 4 weeks are eligible for study enrollment and randomization. Subjects experiencing 3 or more confirmed attacks before the end of the 4-week period may exit the run-in period early and proceed to enrollment and randomization. Subjects who have not had at least 1 confirmed seizure after the 4 week run-in period may have their run-in period extended for an additional 4 weeks, during which time they must have at least 2 confirmed seizures to be eligible for enrollment. study and undergo randomization.

To be eligible for enrollment, subjects who had their run-in period extended must complete the full 8-week run-in period before beginning the treatment period. Subjects who do not meet the minimum seizure frequency requirements during the run-in period, or who are otherwise determined to be ineligible by screening, are considered to have failed the screening and are not permitted to be rescreened for study enrollment.

Treatment period.

After eligibility verification, subjects are randomized 2:1 to receive repeated subcutaneous (SC) administrations of DX-2930 or placebo in a double-blind fashion. Subjects randomized to the DX-2930 group are assigned in a 1:1:1 ratio to receive one of three dosing regimens: 300 mg every 2 weeks, 300 mg every 4 weeks, or 150 mg every 4 weeks. Randomization into all treatment groups is divided into blocks by comparing untreated subjects versus treated subjects (subjects receiving active study drug in protocol DX-2930-02), as well as baseline seizure frequency observed during the run-in period, into the following groups: 1 to <2 attacks in 4 weeks, 2 to <3 attacks in 4 weeks, and >3 attacks in 4 weeks.

Each subject undergoes a treatment period consisting of 13 doses of masked investigational medicinal product (IMP) for 26 weeks from the date of the first dose on Day 0 until two weeks after the last dose. Subjects randomized to one of 4 treatment groups receive either a dose of DX2930 or a dose of placebo, according to the dosage regimen in Table 1. 2.

- 25 045920

table 2

Scheme of administration during the treatment period

Treatment Period Groups by Treatment Option: DX-2930 or Placebo Number of DOZES Dose Day/Week 300 mg every 2 weeks 300 mg every 4 weeks 150 mg every 4 weeks Placebo 1 Day 0/Week 0 DX-2930 DX-2930 DX-2930 Placebo 2 Day 14/Week 2 DX-2930 Placebo Placebo Placebo 3 Day 28/Week 4 DX-2930 DX-2930 DX-2930 Placebo 4 Day 42/Week 6 DX-2930 Placebo Placebo Placebo 5 Day 56/Week 8 DX-2930 DX-2930 DX-2930 Placebo 6 Day 70/Week 10 DX-2930 Placebo Placebo Placebo 7 Day 84/Week 12 DX-2930 DX-2930 DX-2930 Placebo 8 Day 98/Week 14 DX-2930 Placebo Placebo Placebo 9 Day 112/Week 16 DX-2930 DX-2930 DX-2930 Placebo 10 Day 126/Week 18 DX-2930 Placebo Placebo Placebo eleven Day 140/Week 20 DX-2930 DX-2930 DX-2930 Placebo 12 Day 154/Week 22 DX-2930 Placebo Placebo Placebo 13 Day 168/Week 24 DX-2930 DX-2930 DX-2930 Placebo Day 182/Week 26 No dose No dose No dose No dose

Description of treatment.

DX-2930.

DX-2930 is a sterile, preservative-free injection solution, pH 6.0. The active ingredient, DX-2930, is formulated using the following pharmacopoeial components: 30 mM dibasic sodium phosphate dihydrate, 19.6 mM citric acid monohydrate, 50 mM L-histidine, 90 mM sodium chloride, 0.01% Polysorbate 80. Each bottle contains a nominal concentration 150 mg of active ingredient DX-2930 in 1 ml of solution. The study drug is administered by subcutaneous injection into the upper arm in a blinded manner.

For each 300 mg dose of DX-2930, each subject receives a total of 2 mL, divided into 2 separate SC injections of 1.0 mL of DX-2930. 2 Injections are made into the same upper arm, with at least 2 cm between each injection site. For each 150 mg dose of DX-2930, each subject receives a total of 2 mL divided into 2 separate 1.0 mL subcutaneous injections, where one injection is DX-2930 and the other is placebo. 2 Injections are made into the same upper arm, with at least 2 cm between each injection site.

Placebo.

The placebo consists of an inactive composition of the study product: 30 mM dibasic sodium phosphate dihydrate, 19.6 mM citric acid monohydrate, 50 mM L-histidine, 90 mM sodium chloride, pH 6.0 with 0.01% Polysorbate 80. Placebo doses are administered to patients , randomized to placebo treatment, and between doses of DX-2930 for subjects randomized to 300 mg or 150 mg DX2930 every 4 weeks, according to the dosing schedule in Table. 1.

For each placebo dose, each subject receives a total of 2 mL, divided into 2 separate subcutaneous injections of 1.0 mL of placebo. 2 injections are given into the same shoulder, with at least 2 cm between each injection site.

Observation period.

Subjects may additionally undergo safety assessments and additional assessments (ie, pharmacokinetics and pharmacodynamics assessments) during the 8-week observation period. Subjects (or caregivers) are instructed to report any attacks of NAE they have experienced since the last follow-up visit.

Rules for stopping participation in the study.

If at any time it is determined that a dose group must be excluded due to an important safety signal, the remaining unenrolled subjects may be re-randomized into the remaining lower dose DX-2930 or placebo group(s) and continue to participate in the study. double blind. Data for these subjects is used up to

- 26 045920 the moment when the decision is made to reduce the dose in efficacy analyzes and in general in safety analyses.

Dosing for any individual subject should be discontinued if the subject experiences a DX-2930-related serious side effect (or a DX-2930-related clinically significant non-serious side effect) that, in the judgment of the investigator, justifies dosing discontinuation for reasons related to the health of that subject. subject. The researcher has the opportunity to contact the medical supervisor and consult with him on such issues. The subject is followed until completion of all scheduled visits, unless a request is made to discontinue participation in the study. Subjects who are discontinued will not be eligible to participate in the open-label extension (OLE) study.

Study category of patients.

The study is enrolling up to 120 subjects to provide 108 complete subjects. Subjects are 12 years of age or older and have a confirmed diagnosis of HAE (type I or II) and must have experienced at least 1 confirmed attack per 4 weeks during the run-in period. The goal is to enroll at least 5 subjects who are between 12 and 17 years of age. HAE diagnosis is supported by a documented clinical history consistent with HAE and diagnostic testing performed either before or during the screening visit.

Subject inclusion criteria.

Patients who meet the following criteria are subject to the treatment described in the present invention.

1. Men and women 12 years of age or older at the time of screening.

2. Documented diagnosis of HAE (type I or II) based on all of the following:

Documented clinical history consistent with HAE (episodes of subcutaneous or mucosal nonpruritic swelling without associated urticaria).

Diagnostic testing results obtained during screening that confirm HAE type I or II: functional level of C1 inhibitor (C1-INH) < 40% of normal level. Subjects with a functional C1-INH level of 40-50% of normal levels may be included in the study if they also have a C4 level below normal limits. Subjects may begin participating in a run-in period before receiving these diagnostic results. Subjects may be retested if results are inconsistent with clinical history or if they are believed to be confounded by recently used LTP.

At least one of the following: age at first onset of symptoms of angioedema <30 years, family history of HAE Type I or II, or C1q within normal limits.

3. Have a baseline rate of at least 1 investigator-confirmed attack of HAE in 4 weeks, as confirmed during the run-in period.

4. Adult subjects and caregivers of patients under 18 years of age are willing and able to read, understand, and sign the informed consent form. Subjects aged 12–17 years, for whom informed consent is provided by a caregiver, are willing and able to read, understand, and sign the consent form.

5. Men and women who are fertile and sexually active should comply with contraceptive requirements throughout the study period as follows.

Women of childbearing potential must agree to abstain or be advised to use a highly effective form of contraception from the time of screening until 30 days after the final study visit. This includes progestin-only oral contraceptives associated with ovulation inhibition (oral, injectable, or implantable), intrauterine device (IUD, all types), or intrauterine hormone-releasing systems (IUS). A woman whose male partner has had a vasectomy must agree to use another form of medically acceptable contraception. The use of a male condom with or without spermicide or a cervical cap, diaphragm or sponge with spermicide, or a combination (double-barrier methods) is not considered highly effective.

Women without childbearing potential, defined as surgically sterile (post-hysterectomy status, bilateral oophorectomy, or bilateral tubal ligation) or postmenopausal for at least 12 months, did not require contraception during the study.

Men, including men who are surgically sterile (post-vasectomy), with female partners of childbearing potential must agree to be abstinent or use a medically acceptable form of contraception from the time of screening until 60 days after the final study visit.

Subject exclusion criteria.

Patients having one or more of the following criteria may be excluded from the treatment described in the present invention.

1. Concomitant diagnosis of another form of chronic, recurrent angioedema

- 27 045920 edema, such as acquired angioedema (AAE), HAE with normal C1-INH (also known as HAE Type III), idiopathic angioedema or recurrent angioedema associated with urticaria.

2. Use of investigational drug or exposure to investigational device within 4 weeks prior to screening.

3. Exposure to angiotensin-converting enzyme (ACE) inhibitors or any systemically absorbed estrogen-containing medications (such as oral contraceptives or hormone replacement therapy) within 4 weeks before screening.

4. Exposure to androgens (eg, stanozolol, danazol, oxandrolone, methyltestosterone, testosterone) for 2 weeks before entering the run-in period.

5. Use of long-term HAE prophylactic therapy (W-INH, attenuated androgens or antifibrinolytics) for 2 weeks before entering the induction period.

6. Use of short-term HAE prophylaxis for 7 days before entering the introductory period. Short-term prophylaxis is defined as C1-INH, attenuated androgens, or antifibrinolytic agents used to avoid complications of angioedema resulting from indicated medical procedures.

7. Any of the following liver function test abnormalities: alanine aminotransferase (ALT) >3x the upper limit of normal or aspartic aminotransferase (AST) >3x the upper limit of normal or total bilirubin >2x the upper limit of normal (unless the increased bilirubin is the result of Gilbert's syndrome).

8. Pregnancy or breastfeeding.

9. The subject has any condition that may be considered to adversely affect safety or compliance with treatment, interfere with the successful conduct of the study, or interfere with the interpretation of results (for example, a history of substance abuse or dependence, a serious pre-existing medical condition, or other serious comorbidity). pathology that, in the opinion of the investigator, may interfere with the interpretation of the study results).

Primary and secondary endpoints.

The following primary and secondary efficacy endpoints are assessed from day 14 to day 182.

The primary endpoint of the study is the number of HAE attacks.

Secondary endpoints include, in order of significance:

1. number of HAE attacks requiring emergency treatment;

2. number of moderate to severe attacks of HAE.

Efficacy endpoints investigated.

1. Time until the first attack after day 14, i.e. the period during which the subject is seizure-free after day 14 before the first seizure.

2. Number of severe HAE attacks per week; A severe attack of HAE is defined as any attack that has at least one of the following characteristics: severe, resulting in hospitalization (except hospitalization for evaluation <24 hours), hemodynamically significant (systolic blood pressure <90, requires IV hydration, or associated with syncope). condition or condition close to fainting) or laryngeal.

Clinical laboratory tests.

Subjects participating in a clinical trial undergo laboratory tests including general safety parameters (hematology, coagulation, urinalysis and serum chemistry), serology, pregnancy tests, C1-INH functional analysis, C4 analysis, C1q analysis, PK samples, analysis anti-drug antibodies in plasma and PD samples. All laboratory tests are carried out using generally accepted and proven methods.

Example 2: Open-label study to evaluate the efficacy and safety of DX-2930.

Review of the study.

We describe a phase 3, multicenter, randomized, open-label, extension study to evaluate the efficacy and safety of DX-2930, an investigational medicinal product (IMP), in preventing acute attacks in patients with type I and type II HAE who had prior HAE treatment including DX-2930. This study largely follows the previous HAE treatment regimen, which included multiple doses of approximately 150 mg or 300 mg of DX-2930 every four weeks or 300 mg of DX-2930 every two weeks. Typically include subjects (patients recruited from a previous phase of the study, e.g., who were treated with DX-2930 following the treatment regimen disclosed in Example 1, or patients who were not recruited from a previous phase of the study, e.g., who were never treated with DX-2930 2930) aged 12 years or older with a documented diagnosis of HAE Type I or Type II, and who, prior to treatment with DX-2930, experienced at least 1 attack per 4 weeks during the run-in period.

Subjects for this study were 12 years of age or older with a confirmed diagnosis of HAE (Type I or II) and had experienced at least 1 attack in the 4 weeks prior to previous HAE treatment.

- 28 045920 including DX-2930. The diagnosis of HAE is confirmed by a documented medical history confirming HAE and diagnostic testing performed either before or during the screening visit.

Treatment period (i).

Subjects recruited from a previous phase of the study.

Each subject recruited from the previous phase of the study (e.g., patients treated with DX-2930 following the treatment regimens described in the present invention, e.g., Example 1 above) takes one unmasked 300 mg dose of DX-2930 administered subcutaneously (SC ) on day 0. Subjects will not take any additional DX-2930 doses until their first report, and this must be confirmed by the investigator, of an attack of HAE. The time period between the first unmasked dose and the first report of an attack of HAE varied between subjects recruited from the previous phase of the study. Until the subject recruited from the previous phase of the study reports a first episode of HAE, the subject will follow a schedule of visits where the following tests and assessments must be performed: pregnancy test, clinical laboratory tests, physical examination, 12-lead ECG, QoL, PK, PD and collecting samples for anti-drug antibody determination.

Once a subject recruited from a previous phase of the study reports his or her first episode of HAE, the subject will visit the study site to receive a second unmasked dose of DX-2930 as quickly as site schedules allow. At the visit where the second unmasked dose of DX-2930 is administered, the subject undergoes a pre-dose vital signs assessment, physical examination, clinical laboratory tests, and blood samples for PK, PD, and anti-drug antibodies. Vital signs are obtained 1 hour after dosing.

Regardless of when a subject recruited from a previous phase of the study experienced their first attack of HAE, a minimum of 10 days must elapse between the first unmasked dose and the second unmasked dose. After the second dose, the subject recruited from the previous phase of the study will continue to receive repeat SC administrations of an unmasked dose of 300 mg DX-2930 every 2 weeks for the remainder of the treatment period according to the dosing schedule. The treatment period lasts 350 days from the date of the first unmasked dose. The number of doses administered during this period varies among subjects depending on the date of administration of the second dose for each subject, but should not exceed 26 doses.

(ii) Subjects who were not recruited from a previous phase of the study.

Once all screening assessments are completed and subject selection is complete, subjects who were not recruited from the previous phase of the study visit the study site and, after predose assessments, take an unmasked dose of 300 mg DX-2930 administered by SC on day 0. For subjects who do not were recruited from a previous phase of the study, continue SC administration of unmasked doses of 300 mg DX-2930 every 2 weeks throughout the treatment period in accordance with the dosing schedule. A total of 26 doses are administered, with the final dose administered at the study day 350 visit.

(iii) All subjects.

All doses (except the second dose for subjects recruited from the previous phase of the study) require a minimum of 10 days and a maximum of 18 days between administrations, and must fall within the accepted window of ±4 days between study visits. If a subject experiences an acute attack of angioedema at any time during the study that, in the opinion of the investigator, requires medical intervention, standard treatment should be provided based on the subject's medical history and in accordance with locally approved drug information.

DX-2930 administration and study procedures continue without changes to the schedule of activities in accordance with the study protocol, even if the subject is receiving treatment for a breakthrough attack of angioedema on the day of the scheduled dosing of the study drug (in the case of self-administration) or the scheduled study visit.

(iv) Duration of treatment.

All subjects take unmasked doses of DX-2930 during the 350-day treatment period. The number of doses taken by subjects recruited from the previous phase of the study during this period varies among subjects, but should not exceed 26 doses. The final unmasked dose of DX-2930 in these subjects can be administered on day 350 of the scheduled study visit.

Subjects who were not recruited from the previous phase of the study take 300 mg of DX2930 every 2 weeks for a total of 26 doses, with the first dose administered on day 0 and the last dose administered on day 350 at the scheduled study visit.

There is a ±4 day window between scheduled study visits. There should be a minimum of 10 days between any two doses. Except for the interval between the first and second non-mask

- 29 045920 doses for subjects recruited from the previous phase of the study, there is an interval of a maximum of 18 days between any two doses. Subjects are monitored at the study site for 1 hour after dosing at each study scheduled visit. No vital sign monitoring is performed for subjects who elect to self-administer at a location other than the study site.

Investigational drug; dose; and method of administration.

DX-2930 (DX-2930) is a sterile, preservative-free injection solution, pH 6.0. The active ingredient, DX-2930, is formulated using the following pharmacopoeial components: 30 mM dibasic sodium phosphate dihydrate, 19.6 mM citric acid (e.g. , citric acid monohydrate), 50 mM histidine (eg, L-histidine), 90 mM sodium chloride, 0.01% Polysorbate 80. Each vial contained a nominal concentration of 150 mg of the active ingredient DX-2930 in 1 ml of solution. The study drug is administered by subcutaneous injection into the upper arm in a blinded manner.

For each 300 mg dose of DX-2930, each subject receives a total of 2 mL, divided into 2 separate SC injections of 1.0 mL of DX-2930. 2 Injections are made into the same upper arm, with at least 2 cm between each injection site.

For each 150 mg dose of DX-2930, each subject receives a total of 2 mL divided into 2 separate 1.0 mL subcutaneous injections, where one injection is DX-2930 and the other is placebo. 2 Injections are made into the same upper arm, with at least 2 cm between each injection site.

DX-2930 can be self-administered without supervision (parental supervision required for adolescent subjects) after subjects have been trained to do so under the direction of an investigator or designated person and their ability to do so has been confirmed. Subjects are permitted to begin self-administration after receiving the first 2 doses of DX-2930 at the study site and may continue to self-administer all subsequent doses. See also the descriptions in the present invention.

Observation period.

Subjects undergo safety assessments and additional assessments (ie, pharmacokinetics and pharmacodynamics assessments) during an 8-week observation period. Subjects (or caregivers) are instructed that they must inform the center of any attacks of NAE they have experienced since the last follow-up visit.

Rules for stopping participation in the study.

If at any time a dose is determined to have an important safety signal, any dose of DX-2930 may be reduced. Dosing in any individual subject is stopped if the subject experiences a DX-2930-related serious side effect (or a DX-2930-related clinically significant non-serious side effect) that, in the judgment of the investigator, justifies stopping dosing for reasons related to the subject's health. .

Subject inclusion criteria.

Patients who meet the following criteria are subject to the treatment described in the present invention.

1. Men and women 12 years of age or older at the time of screening.

2. Documented diagnosis of HAE (type I or II) based on all of the following.

Documented clinical history consistent with HAE (episodes of subcutaneous or mucosal nonpruritic swelling without associated urticaria).

Diagnostic testing results obtained during screening that confirm HAE Type I or II: Functional C1 inhibitor (C1-INH) level <40% of normal level. Subjects with a functional C1-INH level of 40-50% of normal levels may be included in the study if they also have a C4 level below normal limits. Subjects may begin participating in a run-in period before receiving these diagnostic results. Subjects may be retested if results are inconsistent with clinical history or if they are believed to be confounded by recently used LTP.

At least one of the following: age at first onset of symptoms of angioedema <30 years, family history of HAE Type I or II, or C1q within normal limits.

3. Have a baseline rate of at least 1 investigator-confirmed attack of HAE in 4 weeks, as confirmed during the run-in period.

4. Adult subjects and caregivers of patients under 18 years of age are willing and able to read, understand, and sign the informed consent form. Subjects aged 12–17 years, for whom informed consent is provided by a caregiver, are willing and able to read, understand, and sign the consent form.

5. Men and women who are fertile and sexually active should comply with contraceptive requirements throughout the study period as follows:

Women of childbearing potential must agree to abstinence or are advised to

- 30 045920 use highly effective forms of contraception from the time of screening until 30 days after the final study visit. This includes stable doses, for 3 months before screening, of combined estrogen and progestin-containing oral contraception associated with ovulation inhibition (oral, injectable or implantable), progestin-only oral contraception associated with ovulation inhibition, intrauterine device (IUD, all types ) or intrauterine hormone releasing system (IUS). A woman whose male partner has had a vasectomy must agree to use another form of medically acceptable contraception. The use of a male condom with or without spermicide or a cervical cap, diaphragm or sponge with spermicide, or a combination (double-barrier methods) is not considered highly effective.

Women without childbearing potential, defined as surgically sterile (post-hysterectomy status, bilateral oophorectomy, or bilateral tubal ligation) or postmenopausal for at least 12 months, did not require contraception during the study.

Men, including men who are surgically sterile (post-vasectomy), with female partners of childbearing potential must agree to be abstinent or use a medically acceptable form of contraception from the time of screening until 60 days after the final study visit.

Subject exclusion criteria.

Patients having one or more of the following criteria may be excluded from the treatment described in the present invention.

1. Concomitant diagnosis of another form of chronic, recurrent angioedema, such as acquired angioedema (AAE), HAE with normal C1-INH (also known as HAE Type III), idiopathic angioedema, or recurrent angioedema associated with urticaria.

2. Intake of an investigational drug (other than DX-2930 or other HAE therapies) or exposure to an investigational device within 4 weeks prior to screening.

3. Exposure to angiotensin-converting enzyme (ACE) inhibitors or any estrogen-containing medications with systemic absorption (such as oral contraceptives or hormone replacement therapy) within 4 weeks before screening.

4. Any of the following liver function test abnormalities: alanine aminotransferase (ALT) >3x the upper limit of normal, or aspartic aminotransferase (AST) >3x the upper limit of normal, or total bilirubin >2x the upper limit of normal (unless the increased bilirubin is the result of Gilbert's syndrome) .

5. Pregnancy or breastfeeding.

6. The subject has any condition that may be considered to adversely affect safety or adherence to treatment, interfere with the successful conduct of the study, or interfere with the interpretation of results (for example, a history of substance abuse or dependence, a serious pre-existing medical condition, or other serious comorbidity). pathology that, in the opinion of the investigator, may interfere with the interpretation of the study results).

Prohibited concomitant treatments.

The use of the following treatments will be prohibited during the study.

Long-term prophylaxis (LTP) for HAE (eg, use of C1-INH for long-term prophylaxis, attenuated androgens, or antifibrinolytics) after interruption of LTP (within 3 weeks after the first doses of DX-2930).

Angiotensin-converting enzyme (ACE) inhibitors.

Estrogen-containing medications with systemic absorption (such as oral contraceptives or hormone replacement therapy).

Use of androgens (eg, stanozolol, danazol, oxandrolone, methyltestosterone, testosterone) for non-HAE medical conditions or for HAE after interruption within the first three weeks.

Any other investigational drug or device.

Clinical laboratory tests.

Subjects participating in a clinical trial undergo laboratory tests including general safety parameters (hematology, coagulation, urinalysis and serum chemistry), serology, pregnancy tests, C1-INH functional analysis, C4 analysis, C1q analysis, PK samples, PD samples and analysis of anti-drug antibodies in plasma. All laboratory tests are carried out using generally accepted and proven methods.

Self-administration.

All subjects (adolescents or adults) who are considered suitable candidates (that is, those who have the physical and mental ability to learn and the desire to learn) can self-administer the treatment. Subjects are taught this under the direction of the investigator or designee, and the investigator or designee confirms their understanding during training.

Subjects are allowed to begin self-administration after receiving the first 2 doses of DX-2930 at the study site. Once initiated, subjects are permitted to self-administer subsequent doses of DX-2930 at the study site (when study site visits are scheduled) or at the subject's home or other agreed-upon location (when the study permits non-study site administration). Adolescent subjects self-administering study product will be supervised by a parent/legal guardian/caregiver. Alternatively, the parent/legal guardian/caregiver, after completion of appropriate training, authorizes the adolescent to administer DX-2930 without supervision by study site personnel. Site staff telephone subjects after scheduled self-administrations outside the study site to ensure that administration was completed, collect information on adverse events (AEs), concomitant medications, and ensure that all seizures are properly documented.

Performance assessments.

Additional criteria for efficacy endpoints included.

Time to first attack of HAE in subjects recruited from the previous phase of the study (based on time from first unmasked study dose to first attack of HAE).

Number of investigator-verified HAE attacks during treatment.

Number of investigator-confirmed HAE attacks requiring emergency treatment during the treatment period.

Number of moderate to severe HAE attacks during treatment.

Number of severe attacks of NAE during the treatment period; A severe attack of HAE is defined as any attack that has at least one of the following characteristics: A severe attack of HAE is defined as any attack that has at least one of the following characteristics: severe, resulting in hospitalization (excluding hospitalization for evaluation <24 hours ), hemodynamically significant (systolic blood pressure <90, requires IV hydration, or is associated with syncope or near-syncope), or laryngeal.

Additional studies included:

anti-drug t antibody development, pharmacokinetics (PK), pharmacodynamic (PD) effects, quality of life assessments, DX-2930 injection report, observation of DX-2930 self-administration and subcutaneous injections.

Example 3: Evaluation of HMWK after DX-2930 treatment.

HAE patients were randomized into active drug (DX-2930) groups, including different dose groups (30 mg, 100 mg, 300 mg, and 400 mg) and placebo, and received subcutaneous doses of placebo or DX-2930. 2930 (DX-2930). Plasma samples were collected at predose (predose) and postdose time points on days 8, 22, 50, 64, 92, and 120.

Plasma samples were subjected to biomarker assays to evaluate the pharmacodynamic activity of DX-2930 in patient plasma. Specifically, samples were assessed for the presence of 2-chain HMWK in an assay using an antibody that specifically binds to 2-chain HMWK.

As shown in FIG. 3, administration of DX-2930 resulted in a decrease in 2-chain HMWK levels in patients who took DX-2930, such as 300 mg or 400 mg DX-2930. Levels of 2-chain HMWK in these subjects approached the levels of 2-chain HMWK found in healthy subjects at days 8 and 22 post-administration and decreased further by at least day 92 post-administration.

These results indicate that administration of DX-2930 reduced levels of 2-chain HMWK (a biomarker to assess plasma kallikrein instability and the stabilizing effect of Dx-2930), which was detectable for several months after administration. 2-chain HMWK is also used as a biomarker to evaluate plasma kallikrein.

Other embodiments

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

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

Equivalents

Although the present invention describes and illustrates several embodiments

- 32 045920 of the invention, those skilled in the art will readily imagine many other means and/or structures for performing the function and/or obtaining the results and/or one or more benefits described in the present invention, and each of such variations and/or modifications are considered included within the scope of the embodiments of the present invention presented in the present invention. More typically, those skilled in the art will appreciate that all parameters, dimensions, materials and configurations presented in the present invention are illustrative, and that the exact parameters, dimensions, materials and/or configurations will depend on the particular application for which the guidance is used. described in the present invention. Those skilled in the art will recognize, or be able to discover with no more than routine experimentation, many equivalents to the specific embodiments provided herein. Thus, it is to be understood that the embodiments described above are presented by way of example only, and that, within the scope of the claims and equivalents, embodiments of the invention may be practiced in a manner other than as specifically described and claimed. Embodiments of the present invention relate to each individual feature, system, item, material, kit and/or method presented in the present invention. Furthermore, any combination of two or more such features, systems, items, materials, kits and/or methods is included within the scope of the present invention, as long as such features, systems, items, materials, kits and/or methods are not inconsistent with each other.

It should be understood that all definitions as presented and used in the present invention take precedence over definitions given in dictionaries, definitions in documents incorporated by reference, and/or ordinary meanings of certain terms.

As used herein, the singular terms used in the specification and claims, unless expressly stated otherwise, are to be understood to include at least one.

In the context of the invention, the phrase and/or, as used in the specification and claims, should be understood to mean either or both of the elements so connected, i.e. elements, in some cases present together, and in other cases separately. Many elements listed with and/or should be interpreted in the same way, i.e. one or more elements connected in this way. Optionally, elements other than those specifically identified with the condition may be present and/or whether or not they relate to those specifically identified elements. Thus, as a non-limiting example, a reference to A and/or B, when used in combination with non-limiting phrases such as containing, may in one embodiment refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to A and B (optionally including other elements), etc.

It should be understood that in the context of the invention in the description and claims, or has the same meaning as and/or as defined above. For example, when separating items in a list, the terms either or and/or should be interpreted as inclusive, i.e. including at least one, but also including several of a series or list of items and, optionally, additional items not included in the list. Only terms clearly indicating otherwise, such as only one of or exactly one of or, when used in a claim, consisting of, will refer to the inclusion of exactly one element from a series or list of elements. In general, for purposes of the invention, the term or should be interpreted solely as indicating exclusive alternatives (i.e., one or the other, but not both) when preceded by exclusivity terms such as any, one of, only one of, or exactly one of , consisting essentially of, when used in a claim, shall have its ordinary meaning as used in the field of patent law.

It is to be understood that, as used herein in the specification and claims, the phrase at least one, when referring to one or more elements listed, means at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and without excluding any combinations of elements from the list of elements. This definition also allows that, optionally, there may be elements other than those specifically defined in the list of elements to which the phrase refers, at least whether or not it refers to those specifically defined elements. Thus, by way of non-limiting example, the phrase at least one of A and B (or equivalently at least one of A or B, or equivalently at least one of A and/or B) may refer in one embodiment to at least one, optionally including more than one, A in the absence of B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more, B in the absence of A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless expressly stated otherwise, any methods claimed herein

-

Claims (21)

In a general invention involving multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are stated. In the claims, as well as in the description presented above, all transitional phrases such as containing, including, bearing, having, consisting of, etc. should be understood as non-limiting, i.e. mean including but not limited to. Only the transition phrases consisting of and substantially consisting of must be closed or semi-closed transition phrases, respectively, as specified in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. CLAIM 1. A method for treating exacerbation of hereditary angioedema (HAE) or reducing the frequency of HAE attacks, including: (i) administering to a human subject in need thereof an antibody in a single dose of 300 mg, wherein said human subject has received a first HAE treatment which includes administering said antibody to said subject in multiple doses of 300 mg every two weeks; and (ii) further administering to said human subject the antibody at one or more 300 mg doses if the subject experiences an exacerbation of HAE after (i), wherein said antibody comprises a heavy chain (HC) complementarity determining region (CDR) 1 of SEQ ID NO: 5, HC CDR2 with SEQ ID NO: 6, HC CDR3 with SEQ ID NO: 7 and LC CDR1 with SEQ ID NO: 8, LC CDR2 with SEQ ID NO: 9 and LC CDR3 with SEQ ID NO: 10 ; wherein said human subject has had at least one attack of HAE in the four weeks prior to the first HAE treatment or at least two attacks of HAE in the eight weeks prior to the first HAE treatment. 2. The method of claim 1, wherein in step (ii) the subject is administered multiple doses of 300 mg of antibody every two weeks. 3. The method according to claim 1 or 2, wherein the first dose of step (ii) is administered within one week after the attack of HAE. 4. Method according to any one of claims 1 to 3, wherein the single dose (i) and the first dose (ii) are administered at an interval of at least 10 days. 5. The method of any one of the preceding claims, wherein the antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 3 and a light chain variable domain sequence of SEQ ID NO: 4. 6. The method as claimed in any one of the preceding claims, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof. 7. The method of claim 6, wherein the antibody comprises a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2. 8. The method of any one of the preceding claims, wherein the antibody is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier. 9. The method according to claim 8, where the pharmaceutical composition includes sodium phosphate, citric acid, histidine, sodium chloride and polysorbate 80. 10. The method according to claim 9, where the pharmaceutical composition contains 30 mm sodium phosphate, 19 mm citric acid, 50 mm histidine, 90 mm sodium chloride and 0.01% polysorbate 80, pH 6.0. 11. The method according to any one of the preceding claims, wherein the antibody is administered subcutaneously. 12. The method of any one of the preceding claims, wherein said human subject has HAE or is suspected of having HAE. 13. The method according to any of the preceding claims, wherein said human subject has HAE type I or type II. 14. The method of any one of the preceding claims, wherein said human subject experienced at least two attacks of HAE in the year prior to the first HAE treatment. 15. The method of any one of the preceding claims, wherein said human subject has received one or more previous HAE treatments prior to the first HAE treatment. 16. The method of claim 15, wherein the prior HAE treatment includes a C1 inhibitor (C1-INH), a plasma kallikrein inhibitor, a bradykinin receptor antagonist, an androgen, an antifibrinolytic agent, or a combination thereof. 17. The method of claim 16, wherein the prior treatment for HAE includes C1-INH, ecallantide, icatibant, danazol, tranexamic acid, or a combination thereof. 18. The method according to any one of claims 15 to 17, wherein the subject has undergone a period of tapering during one or more previous HAE treatments. 19. The method according to claim 18, where the period of gradual dose reduction is 2-4 weeks. 20. The method according to any one of claims 15 to 19, wherein the one or more previous HAE treatments are stopped either before the first dose of the antibody in the first HAE treatment or within three weeks after the first dose of the antibody in the first HAE treatment. 21. The method according to any of the preceding paragraphs, where the specified human subject did not have -