Cross-Reference to Related Applications

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/567,406, filed October 3, 2017; U.S. Provisional Application No. 62/516,463, filed June 7, 2017; and U.S. Provisional Application No. 62/469,385, filed March 9, 2017, each of which is incorporated by reference in its entirety.

Statement Regarding Sequence Listing

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is DIAM_037_03WO_ST25.txt. The text file is about 9 KB, was created on March 9, 2018 and is being submitted electronically via EFS-Web. Background

Technical Field

Embodiments of the present disclosure relate to dosage forms of one or more tissue kallikrein-1 ( KLK1) polypeptides which have a total KLK1 polypeptide dosage of about 0.1 μg/kg to about 10.0 μg/kg, including subcutaneous and intravenous dosage forms. Also provided are related devices and methods of use thereof, for example, for treating ischemic and hemorrhagic conditions.

Description of the Related Art

Tissue kallikreins all possess protease activity with a substrate specificity similar to that of trypsin or chymotrypsin. The most well-characterized activity of KLK1 is its enzymatic cleavage of kininogen to produce bradykinin (BK)-like peptides, collectively known as kinins, which activate, either directly or indirectly, subtypes of both bradykinin receptors (BK-B1, BK-B2). Activation of BK receptors by kinins set in motion a large number of complex metabolic pathways in response to ischemia within the body, which can include improved blood flow (through vasodilation), an anti-inflammatory response, cell repair through angiogenesis or vasculogenesis, and decrease of apoptosis.

There is a significant body of scientific studies which show that tissue kallikrein-mediated release increases blood flow in a variety of tissues including kidney and heart (see, e.g., Stone et al., Arterioscler Thromb Vase Biol. 29: 657-664, 2009), and that such is likely one mode by which kallikrein treatment addresses certain conditions. It is therefore believed that KLK1 has the potential to treat a broad spectrum of clinical scenarios where re-establishing blood flow and reducing inflammation in patients is vital to preserving organ function, including brain, kidney, and heart function. However, there remains a need to identify optimal dosage forms and routes of administration that achieve and maintain therapeutic levels of KLKl in humans. The present disclosure address these and other needs.

Brief Summary

Embodiments of the present disclosure relate to the unexpected discovery that formulations of tissue kallikrein-1 (KLKl) have an inverse dose curve, where up to a certain point administration of lower-dosage formulations show an improved pharmacokinetic and/or activity profile relative to higher-dosage formulations.

Certain embodiments therefore include a dosage form comprising one or more tissue kallikrein (KLKl) polypeptides which are formulated at a total KLKl polypeptide dosage of about 1.0 μg/kg to about 5.0 μg/kg or to about 10 μg/kg. In certain embodiments, the dosage form is suitable for subcutaneous or intravenous administration.

In some embodiments, the dosage form comprises a total KLKl polypeptide dosage of about

0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg/kg, including all ranges in between.

Particular dosage forms comprise a total KLKl polypeptide subcutaneous dosage form of about 1.0 to about 4.0 μg/kg, or about 1.0 to about 3.0 μg/kg, or about 1.0 to about 2.0 μg/kg, or about 2.0 to about 5.0 μg/kg, or about 2.0 to about 4.0 μg/kg, or about 2.0 to about 3.0 μg/kg, or about 3.0 to about 5.0 μg/kg, or about 3.0 to about 4.0 μg/kg, or about 2.5 to about 3.5 μg/kg, or about 3 μg/kg.

Particular dosage forms comprise a total KLKl polypeptide intravenous dosage form of about 0.5 μg/kg to about 3.0 μg/kg, or about 0.5 μg/kg to about 2.0 μg/kg, or about 0.5 μg/kg to about 1.0 μg/kg, or about 0.5 μg/kg to about 0.8 μg/kg, or about 0.5 μg/kg to about 0.75 μg/kg, or about 0.75 Some dosage forms comprise a mixture of KLKl glycoforms, for example, a first KLKl polypeptide and a second tissue KLKl polypeptide:

wherein the first KLKl polypeptide has three glycans attached at three different positions per polypeptide and the second KLKl polypeptide has two glycans attached at two different positions per polypeptide; and wherein the first KLKl polypeptide and the second KLKl polypeptide are present in the dosage form at a ratio of about 45:55 to about 55:45.

In some aspects, one or more of the glycans are N-linked glycans. In some aspects, one or more of the glycans are attached at amino acid residues 78, 84, or 141 of KLKl as defined by SEQ ID NO: 3 or 4. In some aspects, the three glycans of the first KLKl polypeptide are N-linked glycans at residues 78, 84, and 141. In some aspects, the two glycans of the second KLKl polypeptide are N- linked glycans at residues 78 and 84 but not 141. In some aspects, the first KLKl polypeptide and the second KLKl polypeptide are present in the dosage form at a ratio of about 50:50.

Some dosage forms comprise a mixture of a triple glycoform of a KLKl polypeptide and a double glycoform of a KLKl polypeptide, wherein the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 45:55 to about 55:45. In some aspects, the triple glycoform includes N-linked glycans at amino acid residues 78, 84, and 141 of KLKl, as defined by SEQ ID NO: 3 or 4. In some aspects, the double glycoform includes N-linked glycans at amino acid residues 78 and 84, but not at amino acid residue 141 of KLKl, as defined by SEQ ID NO: 3 or 4. In some aspects, the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 50:50.

In certain embodiments, the one or more KLKl polypeptide(s) are mature KLKl polypeptides, human KLKl (hKLKl) polypeptides, or mature hKLKl polypeptides, including any combination thereof (for example, SEQ ID NO:3 or 4 and variants thereof).

In some embodiments, the hKLKl polypeptide(s) comprise, consist, or consist essentially of amino acid residues 78-141 of SEQ ID NO:l or amino acids residues 78-141 SEQ ID NO:2, or an active fragment thereof, or an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 78-141 of SEQ ID NO:l or amino acids residues 78-141 SEQ ID NO:2.

In some embodiments, the hKLKl polypeptide(s) comprise, consist, or consist essentially of amino acid residues 25-262 of SEQ ID NO:l or amino acid residues 25-262 of SEQ ID NO:2, or an active fragment thereof, or an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO:l or amino acid residues 25-262 of SEQ ID NO:2.

In some embodiments, the KLKl polypeptide(s) comprise an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO:2, and wherein the KLKl polypeptide(s) comprises E145 and/or A188. In some embodiments, the KLKl polypeptide(s) comprise an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO:2, and wherein the KLKl polypeptide(s) comprises Q145 and/or V188. In some aspects, a dosage form comprises a pharmaceutically acceptable diluent, adjuvant, or carrier. In some aspects, the dosage form is substantially free of other glycosylated isoforms (glycoforms) of KLK1.

In some aspects, the dosage form has endotoxin levels of less than about 1 EU/mg protein, host cell protein of less than about 100 ng/mg total protein, host cell DNA of less than about 10 pg/mg total protein, and/or is substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC).

In some aspects, the dosage comprises a second agent, for example, an agent selected from one or more of an angiotensin receptor blocker, edavarone, finerenone, and bardoxalone, including combinations thereof. In some embodiments, the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof.

Also included are methods of treating a subject in need thereof, comprising administering to the subject a dosage form as described herein. Some embodiments comprise subcutaneously or intravenously administering the dosage form to the subject. Certain methods relate to treating an ischemic condition in the subject, optionally selected from one or more of brain ischemia (ischemic stroke), transient ischemic attack (TIA), cardiac ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and cutaneous ischemia. Particular methods relate to treating vascular dementia. Some methods relate to treating a hemorrhagic condition in the subject, optionally a hemorrhagic stroke, including intracerebral (within the brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke, some methods relate to treating diabetes, for example, type 2 diabetes (T2D). Certain embodiments relate to treating traumatic brain injury (TBI). Some embodiments relate to treating kidney disease, for example, chronic kidney disease, diabetic kidney disease, or polycystic kidney disease. Also included are methods of treating systemic lupus erythematosus (SLE) and related conditions or complications such as lupus nephritis, pulmonary arterial hypertension (PAH), focal segmental glomerulosclerosis, and essential hypertension.

In some embodiments, subcutaneously administering the dosage forms achieves in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides, and in some instances maintains in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration. In some embodiments, intravenously administering the dosage form to the subject achieves in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides, optionally in about or less than about 0.5, 1, 2, 3, or 4 hours following the intravenous administration. In some embodiments, the therapeutically-effective serum level is about 1.0 to about 5.0 ng/ml, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/ml, including all ranges in between, for example, 1.0 to about 5.0 ng/ml, or about 1.0 to about 4.0 ng/ml, or about 1.0 to about 3.0 ng/ml, or about 1.0 to about 2.0 ng/ml, or about 2.0 to about 5.0 ng/ml g, or about 2.0 to about 4.0 ng/ml, or about 2.0 to about 3.0 ng/ml, or about 3.0 to about 5.0 ng/ml, or about 3.0 to about 4.0 ng/ml.

In some embodiments, administration of the dosage form achieves an improved

pharmacokinetic profile or biological effect relative to a higher dosage form, for example, a higher dosage form having a total KLKl polypeptide dosage of at least about 15 μg/kg, or at least about 20 μg/kg, or at least about 50 μg/kg, or at least about 100 μg/kg, or at least about 400 μg/kg or more. In some embodiments, the improved pharmacokinetic profile includes increased serum half-life following a single subcutaneous administration, for example, which is measured at about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration.

Certain embodiments comprise administering the dosage form to the subject as a dosing regimen of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. Specific embodiments include administering the dosage form to the subject as a dosing regimen of about once a day every three days, for instance, by subcutaneous administration.

Certain embodiments comprise intravenously administering one intravenous dosage form to the subject, followed by subcutaneously administering one or more subcutaneous dosages form to the subject, for example, as a dosing regimen of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. In some embodiments, the intravenous administration achieves in the subject a therapeutically-effective serum level of the one or more KLKl polypeptides in about or less than about 0.5, 1, 2, 3, or 4 hours following the intravenous administration, and the subcutaneous administration maintains the therapeutically-effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration.

Certain methods comprise comprising administering a second agent selected from one or more of an angiotensin receptor blocker, edavarone, finerenone, and bardoxalone, including combinations thereof, for example, as part of the same dosage form or as part of a different dosage form or composition. In some embodiments, the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof.

Also included are devices comprising a dosage form as described herein, and which are adapted for or suitable for subcutaneous administration. In some aspects, the device is a syringe. In some aspects, the syringe includes a hypodermic needle assembly attached to the syringe. In some aspects, the syringe includes a protective cover around the needle assembly. In some aspects, the syringe has a needle that is about ½ inch to about 5/8 of an inch in length and has a gauge of about 25 to about 31. Brief Description of the Drawings

Figure 1 shows the results of a KLKl (DM 199) Phase l/l I pilot study in humans for the treatment of Type 2 Diabetes (T2D). Patients (n=37) received placebo or one of two doses (high dose of 15 μg/kg, or low dose of 3 μg/kg) of KLKl every three days for 28 days, and fasting blood glucose (FBG) levels were measured. The high dose (15 μg/kg) showed no significant effect, but the low dose (3 μg/kg) showed a statistically significant (p<0.05) effect on FBG levels relative to baseline. Thus, the lower 3 μg/kg provides an unexpected improvement relative to the higher 15 μg/kg dose.

Figures 2A-2B show that subcutaneous administration of low-dosage (3 μg/kg; n=12) formulations of mature human KLKl glycoforms results in significantly prolonged serum half-file, even relative to intravenous administration of low-dosage formulations (0.75 μg/kg; n=12), in healthy human subjects.

Figure 3 shows the results of a clinical study in which 3 μg/kg dosage of KLKl (DM 199) maintained fairly steady drug levels at the desired or therapeutically-effective serum/plasma concentration of about 3-5 ng/ml. I n contrast, the higher 15/25 μg/kg dosage caused plasma levels to be proportionally higher with greater fluctuation between doses.

Figures 4A-4D shows the results of a KLKl dosage test performed in patients with Type 2 diabetes. Figure 4A provides a summary, Figures 4B and 4D show the results for placebo groups, and Figure 4C shows the results of the (KLKl) DM199 groups. The numbers are a derived measure of insulin resistance ( HOMA2-I R) where higher numbers represent greater insulin resistance and denote greater illness.

Figure 5 shows the results of a 28-day multiple dose study in Type 2 Diabetic patients (n = 12

- 13 per group) on measurements of kidney function (creatinine, blood urea). The low dosage (3 μg/kg) had a significantly greater effect than the higher dosage (15 μg/kg) on improving

measurements of kidney function. Detailed Description

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

Throughout this specification, u nless the context requires otherwise, the words "comprise,"

"comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever follows the phrase

"consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

As used herein, the term "amino acid" is intended to mean both naturally occurring and non- naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally-occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

The terms "endotoxin free" or "substantially endotoxin free" relate generally to dosage forms, compositions, solvents, devices, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo- saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and/or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are typically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art. Also included are methods of producing KLK1 polypeptides in and isolating them from eukaryotic cells such as mammalian cells to reduce, if not eliminate, the risk of endotoxins being present in a composition of the invention. Preferred are methods of producing KLK1 polypeptides in and isolating them from recombinant cells grown in chemically defined, serum free media.

Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Ameobocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/ml, or EU/mg protein. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

The "half-life" of an agent such as KLKl polypeptide of dosage form can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. "Half-life" can also refer to the time it takes for the levels of agent to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and/or any one or more selected tissues.

The terms "modulating" and "altering" include "increasing," "enhancing" or "stimulating," as well as "decreasing" or "reducing," typically in a statistically significant or a physiologically significant amount or degree relative to a control. An "increased," "stimulated" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times {e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount or level produced by a control composition, sample or test subject. A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount or level produced a control composition, sample or test subject. As one non-limiting example, the comparison can be between the amount or level of a pharmacokinetic parameter/profile or biological/therapeutic response produced by administration of a lower-dosage form (e.g., 1-10 μg/kg) of KLKl relative to administration of a higher dosage form of KLKl. Other examples of comparisons and "statistically significant" amounts are described herein.

The terms "polypeptide," "protein" and "peptide" are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. In certain embodiments, the polypeptide is a

"recombinant" polypeptide, produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.

The term "reference sequence" refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

A result is typically referred to as "statistically significant" if it is unlikely to have occurred by chance. The significance level of a test or result relates trad itionally to the amount of evidence required to accept that an event is unlikely to have arisen by chance. In certain cases, statistical significance may be defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true (a decision known as a Type I error, or "false positive

determination"). This decision is often made using the p-value: if the p-value is less than the significance level, then the null hypothesis is rejected. The smaller the p-value, the more significant the result. Bayes factors may also be utilized to determine statistical significance {see Goodman, Ann Intern Med. 130: 1005-13, 1999).

The term "solubility" refers to the property of a KLK1 polypeptide provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 m L), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that ca n dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 6.0, pH 7.0, pH 7.4, pH 8.0 or pH 9.0. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCI (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (for example, pH 6.0) and relatively higher salt (for example, 500m M NaCI and lOmM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (for example, about 20, about 21, about 22, about 23, about 24, or about 25°C) or about body temperature (37°C). In certain embodiments, a KLK1 polypeptide has a solubility of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, or at least about 60 mg/ml at room temperature or at 37°C.

"Substantially" or "essentially" means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

"Treatment" or "treating," as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.

As used herein, the terms "therapeutically effective amount", "therapeutic dose,"

"prophylactically effective amount," or "diagnostically effective amount" is the amount of an agent (e.g., KLK1 polypeptide or dosage form thereof) needed to elicit the desired biological response following administration.

A "subject," as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with a KLK1 polypeptide or a dosage form thereof.

Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances such as host cell proteins or nucleic acids.

A "wild type" or "reference" sequence or the sequence of a "wild type" or "reference" protein/polypeptide may be the reference sequence from which variant polypeptides are derived through the introduction of changes. In general, the "wild type" amino acid sequence for a given protein is the sequence that is most common in nature. Similarly, a "wild type" gene sequence is the polynucleotide sequence for that gene which is most commonly found in nature. Mutations can be introduced into a "wild type" gene (and thus the protein it encodes) either through natural processes or through human induced means.

Each embodiment in this specification is to be applied to every other embodiment unless expressly stated otherwise.

Dosage Forms

Embodiments of the present disclosure relate to dosage forms of one or more tissue kallikrein (KLK1) polypeptides, which are formulated at a total KLK1 polypeptide dosage of about 0.1 μg/kg to about 5 μg/kg or to about 10.0 μg/kg. In some instances, the dosage forms are suitable for (or adapted for) subcutaneous or intravenous administration to a subject, for example, a human subject.

Certain dosage forms comprise, consist, consist essentially of, or are composed of a total KLK1 polypeptide dosage of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg/kg, including all ranges in between.

For instance, certain dosage forms comprise, consist, consist essentially of, or are composed of a total KLK1 polypeptide dosage of about 0.1 μg/kg to about 10.0 μg/kg, about 0.1 μg/kg to about 9.0 μg/kg, about 0.1 μg/kg to about 8.0 μg/kg, about 0.1 μg/kg to about 7.0 μg/kg, about 0.1 μg/kg to about 6.0 μg/kg, about 0.1 μg/kg to about 5.0 μg/kg, or about 0.1 μg/kg to about 4.0 μg/kg, or about 0.1 μg/kg to about 3.0 μg/kg, or about 0.1 μg/kg to about 2.0 μg/kg, or about 0.01 μg/kg to about 1.0 μg/kg, or about 0.5 μg/kg to about 10.0 μg/kg, about 0.5 μg/kg to about 9.0 μg/kg, about 0.5 μg/kg to about 8.0 μg/kg, about 0.5 μg/kg to about 7.0 μg/kg, about 0.5 μg/kg to about 6.0 μg/kg, about 0.5 μg/kg to about 5.0 μg/kg, or about 0.5 μg/kg to about 4.0 μg/kg, or about 0.5 μg/kg to about 3.0 μg/kg, or about 0.5 μg/kg to about 2.0 μg/kg, or about 0.5 μg/kg to about 1.0 μg/kg, or about 1.0 μg/kg to about 10.0 μg/kg, about 1.0 μg/kg to about 9.0 μg/kg, about 1.0 μg/kg to about 8.0 μg/kg, about 1.0 μg/kg to about 7.0 μg/kg, about 1.0 μg/kg to about 6.0 μg/kg, about 1.0 μg/kg to about 5.0 μg/kg, or about 1.0 μg/kg to about 4.0 μg/kg, or about 1.0 μg/kg to about 3.0 μg/kg, or about 1.0 μg/kg to about 2.0 μg/kg, or about 2.0 μg/kg to about 10.0 μg/kg, or about 2.0 μg/kg to about 9.0 μg/kg, or about 2.0 μg/kg to about 8.0 μg/kg, or about 2.0 μg/kg to about 7.0 μg/kg, or about 2.0 μg/kg to about 6.0 μg/kg, or about 2.0 μg/kg to about 5.0 μg/kg, or about 2.0 μg/kg to about 4.0 μg/kg, or about 2.0 μg/kg to about 3.0 μg/kg, or about 3.0 μg/kg to about 10.0 μg/kg, or about 3.0 μg/kg to about 9.0 μg/kg, or about 3.0 μg/kg to about 8.0 μg/kg, or about 3.0 μg/kg to about 7.0 μg/kg, or about 3.0 μg/kg to about 6.0 μg/kg, or about 3.0 μg/kg to about 5.0 μg/kg, or about 3.0 μg/kg to about 4.0 μg/kg, or about 4.0 μg/kg to about 10.0 μg/kg, or about 4.0 μg/kg to about 9.0 μg/kg, or about 4.0 μg/kg to about 8.0 μg/kg, or about 4.0 μg/kg to about 7.0 μg/kg, or about 4.0 μg/kg to about 6.0 μg/kg, or about 4.0 μg/kg to about 5.0 μg/kg, or about 5.0 μ /1< to about 10.0 μg/kg, or about 5.0 μg/kg to about 9.0 μg/kg, or about 5.0 μg/kg to about 8.0 μg/kg, or about 5.0 μ /1< to about 7.0 μg/kg, or about 5.0 μg/kg to about 6.0 μg/kg, or about 6.0 μg/kg to about 10.0 μ /1< , or about 6.0 μg/kg to about 9.0 μg/kg, or about 6.0 μg/kg to about 8.0 μg/kg, or about 6.0 μ /1< to about 7.0 μg/kg, or about 7.0 μg/kg to about 10.0 μg/kg, or about 7.0 μg/kg to about 9.0 μ /1< , or about 7.0 μg/kg to about 8.0 μg/kg, or about 8.0 μg/kg to about 10.0 μg/kg, or about 8.0 μg/kg to about 9.0 μ /1< , or about 9.0 μg/kg to about 10.0 μg/kg. Specific dosage forms have a total KLK polypeptide dosage of about 2.5 μg/kg to about 3.5 μg/kg, or about 3 μg/kg, including dosage forms suitable for subcutaneous administration. Particular dosage forms have a total KLK polypeptide dosage of about 0.5 μg/kg to about 1.0 μg/kg, or about 0.75 μg/kg, including dosage forms suitable for intravenous administration.

Tissue Kallikrein-1 (KLK1) Polypeptides. As noted above, certain dosage forms comprise one or more tissue kallikrein-1 or KLK1 polypeptides. Tissue kallikreins are members of a gene super family of serine proteases comprising at least 15 separate and distinct proteins (named tissue kallikrein 1 through 15) (Yousef et al., 2001, Endocrine Rev; 22: 184-204). Tissue kallikrein-1 is a trypsin-like serine protease. In humans and animal tissues, tissue kallikrein-1 cleaves kininogen into lysyl-bradykinin (also known as kallidin), a decapeptide kinin having physiologic effects similar to those of bradykinin. Bradykinin is a peptide that causes blood vessels to dilate and therefore causes blood pressure to lower. Kallidin is identical to bradykinin with an additional lysine residue added at the N-terminal end and signals through the bradykinin receptor.

The KLK1 gene encodes a single pre-pro-enzyme that is 262 amino acid residues in length and that includes the "pre-" sequence (residues 1-18) and the "pro-" sequence (residues 19-24), which is activated by trypsin-like enzymes. The "mature" and "active" form human KLK1 is a glycoprotein of about 238 amino acid residues (residues 25-262) with a molecular weight of 26 kDa and a theoretical pi of 4.6. KLK1 has five disulfide bonds in its tertiary structure that are believed to be responsible for the protein's high stability, both against trypsin digestion and heat inactivation.

The amino acid sequence of tissue kallikrein-1 is available for a wide variety of species, including, but not limited to, human (SEQ I D NO: l and SWQ I D NO:2), mouse (see, for example, Gen Bank: AAA39349.1, February 1, 1994); domestic cat (see, for example, NCBI Reference Sequence: XP_003997527.1, November 6, 2012); gorilla (see, for example, NCBI Reference Sequence:

XP_004061305.1, December 3, 2012); cattle (see, for example, Gen Bank: AAI51559.1, August 2, 2007); dog (see, for example, CBI Reference Sequence: NP_001003262.1, February 22, 2013); rat (see, for example, GenBank: CAE51906.1, April 25, 2006); and olive baboon (see, for example, NCBI Reference Sequence: XP_003916022.1, September 4, 2012). KLKl is functionally conserved across species in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. A tissue kallikrein-1 polypeptide of the present invention may have any of the known amino acid sequences for KLKl, or a fragment or variant thereof.

In certain embodiments, the KLKl polypeptide is a "mature" KLKl polypeptide. In certain embodiments, the KLKl polypeptide is a human KLKl polypeptide, optionally a mature human KLKl polypeptide. In particular embodiments, the KLKl polypeptide is a recombinant human polypeptide, for example, a recombinant human KLKl polypeptide, optionally in the mature form. Recombinant human KLKl (rhKLKl) can provide certain advantages over other sources of KLKl, such as urinary KLKl (e.g., human KLKl isolated from human urine), including a homogenous preparation of rhKLKl, simpler regulatory path to licensure, and options to alter the amino acid sequence or glycosylation pattern based on cell culture conditions.

Exemplary amino acid sequences of human tissue kallikrein-1 (hKLKl) polypeptides are provided in Table Kl below.

In certain embodiments, a KLKl polypeptide comprises, consists, or consists essentially of SEQ ID NO:l-3 or 4, or residues 1-262, residues 19-262, or residues 25-262 of SEQ ID NO:l or SEQ ID NO:2, including fragments and variants thereof. Amino acids 1 to 18 of SEQ ID NO:l and 2 represent the signal peptide, amino acids 19 to 24 represent propeptide sequences, and amino acids 25 to 262 represent the mature peptide. Thus, the preproprotein includes a presumptive 17-amino acid signal peptide, a 7-amino acid proenzyme fragment and a 238-amino acid mature KLK1 protein.

A comparison between SEQ ID NO:l and SEQ ID NO:2 (or SEQ ID NO:3 and SEQ ID NO:4) shows two amino acid differences between the two hKLKl amino acid sequences. Single-nucleotide polymorphism (SNPs) between the two individuals within a species account for an E to Q substitution at amino acid residue 145 of 262 and an A to V substitution at position 188 of 262. SEQ ID NO:l has an E (glutamic acid) at position 145 and an A (alanine) at position 188, while SEQ ID NO:2 has a Q (glutamine) at position 145 and a V (valine) at position 188. In some embodiments, KLK1 polypeptide has an E at position 145; a Q at position 145; an A at position 188; an A at position 188; an E at position 145 and an A at position 188; a Q at position 145 and a V at position 188; a Q at position 145 and an A at position 188; or an E at position 145 and a V at position 188.

As noted above, certain embodiments include active variants and fragments of reference KLK1 polypeptide. A "variant" of a starting or reference polypeptide is a polypeptide that has an amino acid sequence different from that of the starting or reference polypeptide. Such variants include, for example, deletions from, insertions into, and/or substitutions of residues within the amino acid sequence of the polypeptide of interest. A variant amino acid, in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence. Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. The amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.

In some embodiments, a KLK polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to a reference sequence, such as, for example, an amino acid sequence described herein (for example, SEQ ID NOs: 1-4).

In some aspects, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:l or 3, or to a fragment of SEQ ID NO:l or 3, such as for example, residues 25-262 or residues 78-141 of SEQ ID NO:l. Such a KLK1 polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a V at position 188.

In some aspects, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:2 or 4, or to a fragment of SEQ ID NO:2 or 4, such as for example, residues 25-262 or residues 78-141 of SEQ ID NO:2. Such a KLK1 polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a V at position 188.

"Percent (%) amino acid sequence identity" with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California.

For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

Variants may also include heterologous sequences or chemical modifications which are added to the reference KLK1 polypeptide, for example, to facilitate purification, improve metabolic half-life, or make the polypeptide easier to identify. Examples include affinity tags such as a His-tag, Fc regions, and/or a PEGylation sequence and PEG.

The term "fragment" includes smaller portions of a KLK1 polypeptide (or variants thereof) that retain the activity of a KLK1 polypeptide. Fragments includes, for example, a KLK1 polypeptide fragment that ranges in size from about 20 to about 50, about 20 to about 100, about 20 to about 150, about 20 to about 200, or about 20 to about 250 amino acids in length. In other embodiments, a KLK1 polypeptide fragment ranges in size from about 50 to about 100, about 50 to about 150, about 50 to about 200, or about 50 to about 250 amino acids in length. In other embodiments, a KLK1 polypeptide fragment ranges in size from about 100 to about 150, about 100 to about 200, about 100 to about 250, about 150 to about 175, about 150 to about 200, or about 150 to about 250 amino acids in length. In other illustrative embodiments, a KLK1 polypeptide fragment ranges in size from about 200 to about 250 amino acids in length. Certain embodiments comprise a polypeptide fragment of a full-length KLK1 of about, up to about, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more (e.g., contiguous) amino acid residues. In some embodiments, a fragment may have residues 25-262 or residues 78-141 of a preproprotein sequence. In some embodiments, a fragment may be any such fragment size, as described above, of SEQ D NO:l or SEQ ID NO:2.

In some instances, fragments and variants of a KLK1 polypeptide retain the enzymatic capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. In some embodiments, an active variant or fragment retains serine protease activity of a KLK1 polypeptide that releases kallidin from a higher molecular weight precursor such as kininogen, or that cleaves a substrate similar to kininogen such as D-val- leu-arg-7 amido-4-trifluoromethylcoumarin to release a colorimetric or fluorometric fragment. The protease activity of KLK1 polypeptides can be measured in an enzyme activity assay by measuring either the cleavage of low-molecular- weight kininogen, or the generation of lys-bradykinin. In one assay format, a labeled substrate is reacted with the KLK1 glycoform, and the release of a labeled fragment is detected. One example of such a fluorogenic substrate suitable for KLK1 measurement of activity is D-val- leu-arg-7 amido-4- trifluoromethylcoumarin (D-VLR-AFC, FW 597.6) (Sigma, Cat # V2888 or Ana Spec Inc Cat # 24137). When D-VLR-AFC is hydrolyzed, the free AFC produced in the reaction can be quantified by fluorometric detection (excitation 400 nm, emission 505 nm) or by spectrophotometric detection at 380 nm (extinction coefficient = 12,600 at pH 7.2). Other methods and substrates may also be used to measure KLK1 proteolytic activity.

Glycoforms and Mixtures Thereof. In certain embodiments, the dosage form comprises a mixture of one or more KLK1 polypeptide glycoforms, including dosage forms that comprise defined ratios of double and triple glycosylated KLK1 polypeptides (see U.S. Application No. 14/677,122, incorporated by reference in its entirety).

Human kallikrein has three potential Asn-linked (N-linked) glycosylation sites at residues 78, 84, and 141, relative to the mature amino acid sequence shown, for example, in SEQ ID NO: 3 or 4, as well as putative O-linked glycosylation sites. However, O-linked glycosylation is not detected in naturally-occurring KLK1. By SDS-PAGE analysis, KLK1 polypeptides glycosylated at all three positions (positions 78, 84, and 141) are detected as the high molecular weight band and are referred to herein as the high-molecular weight, triple glycosylated glycoform of KLK1 (or "high glycoform" or "triple glycoform" KLKl). By SDS-PAGE analysis, KLKl polypeptides glycosylated at only two of three available positions (positions 78 and 84) are detected as a low molecular weight band and are referred to herein as the low-molecular weight, double glycosylated glycoform of KLKl (or as "low glycoform" or "double glycoform" KLKl).

Certain dosage forms therefore comprise a mixture of KLKl glycoforms at a defined ratio, for example, comprising a first KLKl polypeptide and a second KLKl polypeptide, wherein the first KLKl polypeptide has three glycans attached at the three different positions available for glycosylation in the polypeptide, and wherein the second KLKl polypeptide has two glycans attached at only two of the three different positions available for glycosylation in the polypeptide. In certain embodiments, the first and second KLKl polypeptides are present in the dosage form at a ratio of about 45:55 to about 55:45, including, for example, about 46:54, about 47:53, about 48:52, about 49:51, about 51:49, about 52:48, about 53:47, and about 54:46, including all integers and decimal points in between. In specific embodiments, the first and second KLKl polypeptides are present in the dosage form at a ratio of about 50:50. In some embodiments, the ratio of the first and second KLKl polypeptides is not about 60:40. In some embodiments, the ratio of the first and second KLKl polypeptides is not about 40:60. In certain embodiments, the dosage form is free or substantially free of other glycosylated isoforms (glycoforms) of KLKl.

Some dosage forms comprise a triple glycoform of a KLKl polypeptide and a double glycoform of a KLKl polypeptide, wherein the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 45:55 to about 55:45 including, for example, about 46:54, about 47:53, about 48:52, about 49:51, about 51:49, about 52:48, about 53:47, and about 54:46. In some embodiments, the triple glycoform and the double glycoform are present in the dosage form at a ratio of about 50:50. In some embodiments, the ratio of the triple glycoform and double glycoform is not about 60:40. In some embodiments, the ratio of the triple glycoform and double glycoform is not about 40:60. In certain embodiments, the dosage form is free or substantially free of other glycosylated isoforms (glycoforms) of KLKl.

The ratios of the double and triple glycosylated isoforms of KLKl can be detected and quantitated by a variety of methods, including high performance liquid chromatography (HPLC), which may include reversed phase (RP-HPLC), lectin affinity chromatography and lectin affinity electrophoresis. The preparation and characterization of KLKl glycoform mixtures is described in U.S. Application No. 14/677,122, incorporated by reference in its entirety.

Additional Agents. In certain embodiments, the dosage form comprises one more additional therapeutic agents, for example, a second therapeutic agent. In some embodiments, the additional agent is selected from one or more of an angiotensin receptor blocker, edavarone, finerenone, and bardoxalone, including combinations thereof. Examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof.

Purity. In some embodiments, the "purity" of a dosage form is characterized, for example, by the amount (e.g., total amount, relative amount, percentage) of host cell protein(s), host cell DNA, endotoxin, and/or percentage single peak purity by SEC HPLC. In some instances, the purity of a dosage form is characterized by the amount (e.g., percentage) of KLK1 polypeptide relative to other components, for example, any one or more of the foregoing.

In some embodiments, purity of a dosage form is characterized relative to or by the levels or amount of host cell proteins. The host cells used for recombinant expression may range from bacteria and yeast to cell lines derived from mammalian or insect species. The cells contain hundreds to thousands of host cell proteins (HCPs) and other biomolecules that could contaminate the final product. The HCP may be secreted along with the protein of interest, or released by accidental lysing of the cells, and may contaminate the protein of interest. Two types of immunological methods may be applied to HCP analysis: Western blotting (WB) and immunoassay (IA), which includes techniques such as ELISA and sandwich immunoassay or similar methods using radioactive, luminescent, or fluorescent reporting labels. Compositions of the present invention may include host cell protein of less than about 500, less than about 400, less than about 300, less than about 200, less than about 100 or less than about 50 ng/mg total protein.

In some instances, purity is characterized relative to or by the levels or amount of host cell

DNA. Detection of residual host cell DNA may be performed by Polymerase Chain Reaction (PCR) with a variety of primers for sequences in the host cell genome. Residual host cell DNA is generally reported as being below a certain threshold level, but may also be quantitated with a rPCR method. Compositions of the present invention may include host cell deoxyribonucleic acid (DNA) of less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 10 pg/mg total protein.

In certain embodiments, purity is characterized relative to or by the amount or levels of endotoxin. As noted herein, endotoxin is extremely potent, heat stable, passes sterilizing membrane filters, and is present everywhere bacteria are or have been present. An Endotoxin Unit (EU) is a unit of biological activity of the USP Reference Endotoxin Standard.

The bacterial endotoxins test (BET) is a test to detect or quantify endotoxins from Gram- negative bacteria using amoebocyte lysate (white blood cells) from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Limulus amoebocyte lysate (LAL) reagent, FDA approved, is used for all USP endotoxin tests. There are at least three methods for this test: Method A, the gel-clot technique, which is based on gel formation; Method B, the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and Method C, the chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex.

At least two types of endotoxin tests are described in the USP <85> BET. Photometric tests require a spectrophotometer, endotoxin-specific software and printout capability. The simplest photometric system is a handheld unit employing a single-use LAL cartridge that contains dried, pre- calibrated reagents; there is no need for liquid reagents or standards. The FDA-approved unit is marketed under the name of Endosafe^-PTS™. The device requires about 15 minutes to analyze small amounts of sample, a 25 μΐ aliquot from CSP diluted in a sterile tube, and to print out results. In contrast, gel-clot methods require a dry-heat block, calibrated pipettes and thermometer, vortex mixer, freeze-dried LAL reagents, LAL Reagent Water (LRW) for hydrating reagents and

depyrogenated glassware. In this clot test, diluted sample and liquid reagents require about an hour for sample and positive- control preparation and an hour's incubation in a heat block; results are recorded manually. Thus, the simplicity and speed of the automated system make it ideally suited to the pharmacy setting.

In some instances, the purity of a dosage form is characterized by the degree of aggregation. For instance, the degree of aggregation of KLK1 can be determined by Size-exclusion chromatography (SEC), which separates particles on the basis of size. It is a generally accepted method for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. Certain compositions are also substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC). Certain embodiments are free of aggregates with greater than about 96%, about 97%, about 98%, or about 99%, appearing as a single peak by SEC HPLC.

In certain embodiments, the "purity" of the KLK1 polypeptide(s) in a dosage form is specifically defined. For instance, certain dosage forms comprise one or more hKLKl polypeptides that are at least about 80, at least about 85, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, or 100% pure, including all decimals in between, relative to other components in the dosage form. Purity can be measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

In certain embodiments, the dosage form has one or more of the following determinations of purity: less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC HPLC.

In some instances, the dosage forms are formulated with pharmaceutically acceptable excipients, diluents, adjuvants, or carriers, for instance, to optimize stability and achieve isotonicity. In certain aspects, the pH of the dosage form is near physiological pH or about pH 7.4, including about pH 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5, or any range thereof. In some embodiments, a dosage form comprises a KLK1 polypeptide in combination with a physiologically acceptable carrier. Such carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., Edition 21 (2005).

The phrase "physiologically-acceptable" or "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce a significant allergic or similar untoward reaction when administered to a human. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparations can also be emulsified.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

The dosage forms described herein may be formulated for administered by a variety of techniques, including, for example, subcutaneous and intravenous administration. Particular embodiments include administration by subcutaneous injection. In some instances, a subcutaneous injection (abbreviated as SC, SQ, sub-cu, sub-Q or subcut with SQ) is administered as a bolus into the subcutis, the layer of skin directly below the dermis and epidermis, collectively referred to as the cutis. Exemplary places on the body where people can inject SC most easily include, without limitation, the outer area of the upper arm, just above and below the waist, excepting in certain aspects the area right around the navel (a ~ 2-inch circle), the upper area of the buttock, just behind the hip bone, and the front of the thigh, midway to the outer side, about 4 inches below the top of the thigh to about 4 inches above the knee. These areas can vary with the size of the person. Also, changing the injection site can prevent lumps or small dents called lipodystrophies from forming in the skin.

Subcutaneous injections usually go into the fatty tissue below the skin and in certain instances can utilize a smaller, shorter needle. In specific instances, a needle that is about ½ inch to about 5/8 of an inch in length with a gauge of about 25 to about 31 is sufficient to subcutaneously administer the medication. As will be appreciated by someone skilled in the art, these are general recommendations and SC injections may be administered with needles of other sizes. In some embodiments SC administration is performed by pinching-up on the tissue to prevent injection into the muscle, and/or insertion of the needle at a ~ 45° angle to the skin.

Also included are methods of treating a subject in need thereof, comprising administering to the subject an effective amount of a dosage form as described herein. For instance, certain embodiments include methods of treating an ischemic condition, vascular dementia, a hemorrhagic condition, traumatic brain injury (TBI), diabetes, or kidney disease, among others.

Thus, in some embodiments, the subject has an ischemic condition. Non-limiting examples include brain ischemia (ischemic stroke), transient ischemic attack (TIA), cardiac ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and cutaneous ischemia. In some embodiments, the subject has vascular dementia. In some embodiments, the subject has a hemorrhagic condition, for example, a hemorrhagic stroke, including intracerebral (within the brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. In some embodiments, the subject has diabetes, for example, type 2 diabetes (T2D). In particular embodiments, the subject has a traumatic brain injury (TBI). In some

embodiments, the subject has a kidney disease, for example, chronic kidney disease, diabetic kidney disease, or polycystic kidney disease. In some embodiments, the subject has systemic lupus erythematosus (SLE) or a related condition or complication such as lupus nephritis. In particular embodiments, the subject has pulmonary arterial hypertension (PAH), focal segmental

glomerulosclerosis, or essential hypertension. These and related medical conditions can be diagnosed according to routine techniques in the art.

In certain instances, administration of the dosage form achieves in the subject a

therapeutically-effective serum level of the one or more KLK1 polypeptides. In some instances, administration of the dosage form achieves a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours following administration. In some instances, the dosage form is administered intravenously or subcutaneously. In some instances, the therapeutically-effective serum level is about or at least about 1.0 to about or at least about 5.0 ng/ml, or about or at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/ml, including all ranges in between.

In some instances, administration of the dosage form achieves and maintains in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides. For instance, in some embodiments, administration of the dosage forms achieves a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, and maintains in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides for about or at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the administration (e.g., a single subcutaneous or intravenous administration). In some instances, the therapeutically-effective serum level is about or at least about 1.0 to about or at least about 5.0 ng/ml, or about or at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/ml, including all ranges in between.

In some instances, administration of the dosage form achieves or results in an improved pharmacokinetic profile or biological (e.g., therapeutic) effect relative to a higher KLK1 polypeptide dosage form. For example, in some instances, subcutaneous administration of the dosage form achieves an improved pharmacokinetic profile or biological (e.g., therapeutic) effect relative to a dosage form having a total KLK1 polypeptide dosage of at least about 15 μg/kg, at least about 20 μg/kg, or at least about 50 μg/kg, or at least about 100 μg/kg, or at least about 400 μg/kg, or more. In some instances, the improved pharmacokinetic profile includes increased serum half-life following a single subcutaneous or intravenous administration, which is measured, for example, at about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration (e.g., a single subcutaneous administration).

Certain embodiments include a dosage regimen of administering one or more KLK1 dosage forms at defined intervals over a period of time. For example, certain dosage regimens include administering a KLK1 dosage form once or twice a day, once or twice every two days (e.g., once a day every other day), once or twice every three days (e.g., once a day every third day following an initial or earlier administration), once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week, once or twice every other week. Specific dosage regimens include administering a KLK1 dosage form once a day every three days (e.g., once a day every third day following an initial or earlier administration), including wherein the dosage form is administered subcutaneously. Specific embodiments include intravenously administering at least one intravenous dosage form to the subject, followed by subcutaneously administering one or more subcutaneous dosages form to the subject, for example, as a dosing regimen of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. In particular embodiments, the intravenous administration or dosage form achieves in the subject a therapeutically-effective serum level of the one or more KLK1 polypeptides in about or less than about 0.5, 1, 2, 3, or 4 hours following the intravenous administration, and the subcutaneous administration or dosage form maintains the therapeutically-effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, following the subcutaneous administration.

Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. In some instances, preparation are substantially endotoxin-free or pyrogen-free, as described herein.

According to the FDA Guidance for Industry; Estimating the Maximum Safe Starting Dose in Initial Clinical Trial for Therapeutics in Adult Healthy Volunteers (July 2005), Appendix D: Converting animal doses to human equivalent doses. A human equivalent dose is 1/7 the rat dose and a human equivalent dose is 1/12 a mouse dose.

In some embodiments, a dosage form describe herein is administered with one or more additional therapeutic agents or modalities. In some aspects, administration of the dosage form allows for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic modalities alone, providing relief from the toxicity observed with the administration of higher doses of the other modalities. One or more additional therapeutic agents may be administered before, after, and/or coincident (e.g., together with) to the

administration of a dosage form described herein. A dosage form and any additional therapeutic agents can be administered separately or as part of the same mixture or cocktail. As used herein, an additional therapeutic agent includes, for example, an agent whose use for the treatment of a condition (e.g., an ischemic or hemorrhagic condition) is known to persons skilled in the art. Examples of additional agents include angiotensin receptor blockers, edavarone, finerenone, and bardoxalone, including combinations thereof. Particular examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan, including combinations thereof. Devices. Also included are devices that comprise a dosage form described herein, including devices suitable for subcutaneous or intravenous delivery. In some embodiments, the device is a syringe. In some embodiments, the syringe is attached to a hypodermic needle assembly, optionally comprising a protective cover around the needle assembly. In some embodiments, the needle may be about ½ inch to about 5/8 of an inch in length and has a gauge of about 25 to about 31. Certain embodiments thus include devices that attached or attachable to a needle assembly that is suitable for subcutaneous administration, comprising a dosage form described herein. For example, certain devices include a vial or syringe, optionally where the vial or syringe is attachable to or is attached to a hypodermic needle assembly. Also included are vials having a rubber cap, where a needle/syringe can be inserted into the vial via the rubber cap to withdraw the dosage form for subcutaneous administration.

In particular aspects, the device is a syringe that is attachable or attached to a hypodermic needle, and is packaged with one or more removable and/or permanent protective covers around the needle or needle assembly. For instance, a first removable protective cover (which is removed during administration) can protect a user or other person from the needle prior to administration, and a second protective cover can be put {i.e., snapped) into place for safe disposal of the device after administration.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Examples

Example 1

Pharmacokinetics of Low-Dosage KLK1 Formulations in Humans The pharmacokinetics of low-dosage KLK1 formulations were evaluate in humans. The formulations are composed of mixture of double and triple glycosylation isoforms of mature human KLK1, which were prepared as described in U.S. Application No. 14/677,122 (incorporated by reference).

A single dose comparison study was designed to establish safety, tolerability and pharmacokinetics of the KLK1 formulation after 30 minute IV infusion and a single subcutaneous injection. The IV dose was 0.75 μg/kg and the subcutaneous dose was 3 μg/kg. Study groups consisted of 12 volunteers with 6 women and 6 men in each.

The intravenous dose was supplied to the clinical site as a 104.4 mg/ml drug product dissolved in phosphate-buffered saline (pH = 7.2). The volume of KLK1 polypeptide required for dosing was calculated based on the body weight of each study volunteer and the dose level. The subcutaneous dose was provided as a 26.1 mg/ml solution in phosphate-buffered saline. Prior to administration this dosage was diluted to 2.61 mg/ml in normal saline and the volume of injection adjusted according to the body weight of the study participant.

The concentration of KLKl in plasma was measured by ELISA using a KLKl-specific antibody.

This method has been developed and validated for use in human clinical trials.

As shown in Figures 2A-2B, subcutaneous administration of low-dosage (3 μg/kg; n=12) formulations of mature human KLKl glycoforms not only achieved effective plasma levels of KLKl, but also resulted in significantly prolonged serum half-file, even relative to intravenous administration of low-dosage formulations (0.75 μg/kg; n=12), in healthy human subjects. Intravenous administration of low-dosage formulations of mature human KLKl glycoforms rapidly achieved effective plasma levels of KLKl. Figure 2A shows the serum levels at 80 hours and Figure 2B shows serum levels at 4 hours post-administration.

The pharmacokinetic (PK) profile of two KLKl subcutaneous dosing strategies (3 μg/kg; and 15/25 μg/kg) in healthy volunteers was also evaluated in an phase I clinical study. The results are shown in Figure 3. The points represent the mean values derived from six participants per group. The arrows represent times of dosing. The 3 μg/kg dose, which is considered the target dose from the most recent patent, maintained fairly steady drug levels at the desired or therapeutically-effective serum/plasma concentration of about 3-5 ng/ml. In contrast, the higher dosage caused levels to be proportionally higher with a greater fluctuation between doses.

A dosage test of KLKl was performed in patients with Type 2 diabetes. The results are shown in Figures 4A-4D. The points are results of a meal tolerance test conducted ~2 hours after dosing, using the HOMA2-I R measure of insulin resistance three hours after dosing. The numbers are a derived measure of insulin resistance where higher numbers represent greater insulin resistance and denote greater illness. Figure 4D shows the results from the placebo group and the Figure 4C shows the results of the drug groups (DM199). The Day 1 test was run as a baseline followed by single doses of the indicated dose level on subsequent days. The insulin resistance improved on Day 5 when a low dose was tested compared to the results of Day 8 when a higher dose was tested (Figure 4C), suggesting that the low dosage was more effective.

A 28-day multiple dose study in Type 2 Diabetic patients (n = 12 - 13 per group) was also performed. The results are shown in Figures 5A-5B. The bars represent the mean ± SEM change from baseline serum creatinine and urea concentration. These are typical measures of kidney function where above normal values indicate kidney impairment. This result indicates that the low dose (3 μg/kg) had a significantly greater effect than the higher dose on improving measurements of kidney function. The P values are based on comparisons between the 3 μg/kg group and placebo.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.