Fusion protein inhibitor of KLK5

Abstract

Among other things, polypeptides are disclosed that inhibit or suppress the activity of kallikrein-related proteases and are therefore useful for treating diseases and conditions associated with dysregulated kallikrein-related protease activity. Pharmaceutical compositions and methods of treatment using the polypeptides and pharmaceutical compositions are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 390,780, filed July 20, 2022. [Background technology]

[0002] Tissue kallikreins (KLKs) are a family of 15 trypsin- and chymotrypsin-like serine proteases. KLKs are secreted as proenzymes and require specific amino-terminal proteolysis to remove terminal peptide moieties for activation. While some KLKs depend on activation by other KLKs or other proteases, some KLKs, such as KLK5, are capable of self-activation. Thus, KLKs function through proteolytic cascades in the body. KLKs are widely expressed in diverse tissues, including the kidney, brain, respiratory tract, gastrointestinal tract, epidermis, and reproductive organs. KLKs generally regulate several essential physiological functions in a cell-specific manner, including regulation of immunity, inflammation, and carcinogenesis.

[0003] In addition to their other roles, KLKs play an important role in the epidermis. KLK5 and KLK7 degrade desmosomal proteins, a group of proteins responsible for the structural integrity of the epidermis. For example, desmosome degradation, a process known as desquamation, is required for the shedding of dead cells from the skin surface and maintaining a healthy epidermis (Simon et al. 2001; Caubet et al. 2004). As such, KLKs play a role in maintaining the integrity of the skin barrier and preventing desquamation and inflammation. KLK5 is a key regulator of proteolytic activity in the epidermis, as it can activate not only its own proenzyme but also many other KLK family proenzymes, and has been shown to activate pro-KLK7 (Caubet et al. 2004; Brattsand et al. 2005; Yoon et al. 2007). KLK5 has also been shown to activate the metalloproteinases meprin-α and meprin-β, which are involved in basal keratinocyte proliferation and differentiation processes (Ohler et al. 2010). In addition, KLK5 and KLK14 can activate proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor present on the membranes of many cell types, including keratinocytes, and play a regulatory role in inflamed skin (including the generation of a proinflammatory environment, activation of Langerhans cells, and production of proinflammatory cytokines leading to the induction of allergic Th2 cells), epidermal barrier function, and pruritus (Oikonomopoulou et al. 2006; Stefansson et al. 2008). KLKs, including KLK5, are also involved in processing cathelicidin antimicrobial peptides that kill microorganisms and modify host immune and cell growth responses (Lai and Gallo, 2009; Yamasaki et al. 2006; Eissa et al. 2011).

[0004] Several skin disorders are characterized by elevated protease activity (Komatsu et al. 2002, 2008; Descargues et al. 2006; Yamasaki et al. 2007; Cork et al. 2009). Increased KLK5 activity can be observed in skin samples from rosacea patients, who exhibit abnormally high levels of cathelicidin in facial skin (Yamasaki et al. 2007). Netherton syndrome is a severe autosomal recessive disorder also associated with increased KLK5 activity, characterized by congenital ichthyosis with defective keratinization, specific hair shaft defects (bamboo hair), and severe atopic symptoms, including atopic dermatitis and hay fever (Netherton, 1958; Burns et al. 2004).

[0005] However, treatments for diseases and conditions associated with increased KLK activity are largely nonspecific, targeting symptoms rather than the underlying cause of the disease, and resulting in undesirable side effects, such as atrophy after long-term use of glucocorticoids. Thus, there is a need to develop additional KLK inhibitors, particularly KLK5 inhibitors, that have therapeutic potential in the treatment of numerous diseases and conditions. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, the disclosure provides a SPINK9 polypeptide comprising (e.g., at least one) mutant SPINK9 domain. In some embodiments, the at least one mutant SPINK9 domain comprises an amino acid sequence represented by Formula I: Ile Glu Cys Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro XI Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly Ser Asp Gly Lys Thr Tyr X8 Asn Asp Cys Phe Phe Cys Ser Lys Val Lys Lys Thr Asp Gly Thr Leu Lys Phe Val His Phe Gly Lys Cys (SEQ ID NO: 3), where X1-X8 are as defined herein.

[0007] In some embodiments, the at least one mutant SPINK9 domain comprises an amino acid sequence represented by Formula III, wherein X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro X1Gln X2X3X4Cys X5X6X7Tyr Asp Pro Ile Cys Gly Ser Asp Gly Lys Thr Tyr 17 is as defined herein.

[0008] In some embodiments, the at least one mutant SPINK9 domain comprises an amino acid sequence represented by Formula IV, wherein X9X 10 X11 Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro X1Gln Cys (SEQ ID NO: 136), in the formula, X1~X 11 is as defined herein.

[0009] Preferably, the amino acid sequences represented by Formula I, Formula III, and Formula IV are not the wild-type sequence of the SPINK9 domain (ie, SEQ ID NO: 2).

[0010] In further aspects, the disclosure provides compositions, including pharmaceutical compositions, comprising SPINK9 polypeptides, genetic constructs comprising nucleic acid sequences encoding SPINK9 polypeptides, host cells comprising such genetic constructs, and methods for treating diseases and conditions using the SPINK9 polypeptides and pharmaceutical compositions thereof. Such SPINK9 polypeptides can be used to inhibit one or more KLKs, such as KLK5. [Brief explanation of the drawings]

[0011] [Figure 1] The average body weights of animals administered representative mutant SPINK9 polypeptides are shown. [Figure 2] Plasma concentrations of representative mutant SPINK9 polypeptides using an Fc+Fc ELISA method are shown. [Figure 3] 1 shows the plasma concentrations of representative mutant SPINK9 polypeptides using the KLK5+Fc ELISA method. DETAILED DESCRIPTION OF THE INVENTION

[0012] KLKs and their specific inhibitors play an important role in regulating proteolytic activity, particularly in the skin. Alterations in this balance, resulting in dysregulation of KLK expression and / or activity, are involved in several human diseases and conditions (Paliouras, M, Biol Chem, 2006, Vol. 387, pages 643-652). In particular, the serine protease KLK5 and the serine protease inhibitor SPINK5 have each been shown to be involved in regulating epithelial desquamation. The SPINK5 gene encodes the 15-domain protein serine protease inhibitor Kazal type 5 (SPINK5, also known as lymphoepithelial Kazal type-related inhibitor, LEKTI).

[0013] SPINK5 is synthesized as three distinct high-molecular-weight precursors containing up to 15 inhibitory domains, represented by Kazal-like domain arrangements. The precursors are rapidly processed into shorter fragments and secreted extracellularly. Some of these shorter fragments have been shown to inhibit KLK5 activity. The inhibitory effect of wild-type SPINK5 and related KLKs is pH-dependent, with loss of KLK inhibition at acidic pH, similar to that of the outer layers of skin. Loss of function mutations in SPINK5 lead to the absence of KLK5 inhibitory activity and hyperactivation of KLK5.

[0014] Loss of function in SPINK5 mutations and the resulting increased activity of serine proteases (e.g., KLK5) have been shown to be causative factors in various diseases, such as Netherton syndrome and autosomal recessive ichthyosis with hypotrichosis. For example, Netherton syndrome is associated with mutations in the SPINK5 gene, indicating that serine proteases are indeed important for skin physiology (Chavanas et al., 2000). Netherton syndrome is characterized by congenital ichthyosis-like erythroderma, hair loss, and atopic symptoms (Comel, 1949; Netherton, 1958; Chavanas et al., 2000). Severe complications are also associated with Netherton syndrome, including recurrent bacterial infections, hypernatremic dehydration, and growth retardation.

[0015] Serine protease inhibitor Kazal type 9 (SPINK9) is a 7.7 kDa serine protease inhibitor related to SPINK5 and is expressed almost exclusively in areas where the stratum corneum is thickened or hyperkeratosis occurs, such as the upper layers of the palmoplantar epidermis and corns (Brattsand et al. 2009, Meyer-Hoffert et al. 2009). Unlike SPINK5, SPINK9 contains a single inhibitor domain that has been shown to be a KLK5-specific inhibitor. Notably, the two areas of the body that are not affected by congenital SPINK5 mutations in Netherton syndrome are the areas where SPINK9 is expressed: the soles of the feet and the palms of the hands. Thus, SPINK9 also plays a role in regulating skin physiology.

[0016] As disclosed herein, SPINK9 polypeptides, as well as modifications and variants thereof, can be utilized for their interaction with KLK proteases (e.g., KLK5). Accordingly, provided herein are polypeptides and compositions thereof that target enzymes (e.g., KLK family proteases) associated with diseases, particularly skin disorders, as well as methods of treatment using such polypeptides and compositions thereof. Such polypeptides and compositions thereof are useful for treating or preventing diseases and conditions characterized by aberrant KLK activity (e.g., aberrant KLK5 activity).

[0017] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0018] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, "Principles of Neural Science," McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics," Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", W.H. Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", W.H. Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0019] Chemical terms used herein, unless otherwise defined herein, are used in accordance with conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms," Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0020] All patent applications, patents, and publications cited herein are incorporated herein by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and except to the extent that the incorporated material contradicts the express disclosure of this specification, in which case the language of this disclosure will control.

[0021] As used herein, the terms "administering," "administering," or "administration of" refer to the introduction of an agent, including a therapeutic agent, into a subject and can be performed using one of a variety of methods known to those of skill in the art. For example, administering can be performed, e.g., intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, topically, and transdermally (by absorption). Administration can also be performed by rechargeable or biodegradable polymeric or other devices, e.g., patches and pumps, or formulations that provide extended, delayed, or controlled release of the compound or agent. Administration can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0022] As used herein, the term "biologically active fragment" refers to a fragment of a polypeptide that still contains the specific biological activity of the parent polypeptide. For example, in the context of the present disclosure, a "biologically active fragment" of SPINK9 is a fragment of SPINK9 containing a mutant SPINK9 domain that is capable of inhibiting or suppressing the activity of a KLK (e.g., KLK5), as determined using the method described in Example 2 herein. In some embodiments, a "biologically active fragment" of SPINK9 is a fragment of SPINK9 containing a mutant SPINK9 domain that is capable of inhibiting or suppressing the activity of a KLK (e.g., KLK5) by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, compared to wild-type SPINK9 (e.g., SPINK9 having the sequence of SEQ ID NO: 1 or 2).

[0023] As used herein, the phrase "co-administration" or "co-administered" refers to any form of administration of two or more different drugs, such that a second drug is administered while a previously administered drug is still effective in the body (e.g., two drugs are effective in a patient simultaneously, which may include a synergistic effect of the two drugs). For example, different drugs can be administered either simultaneously or sequentially, in either the same formulation or in separate formulations. Thus, a patient receiving such treatment can benefit from the combined effects of the different drugs.

[0024] The phrase "consisting essentially of," as used herein to refer to compounds and compositions, means that certain additional components may be present in such a compound or composition, i.e., those that do not substantially affect the essential characteristics of the compound or composition (e.g., the inhibitory activity of a SPINK9 polypeptide or the inhibitory activity of a mutant SPINK9 domain).

[0025] As used herein, the term "inhibit" means to reduce by an objectively measurable amount or degree. In various embodiments, "inhibit" means to reduce by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent compared to a relevant control. In one embodiment, "inhibit" means to reduce by 100 percent, i.e., to stop or eliminate.

[0026] The term "variant," as used herein, broadly refers to a polypeptide that contains one or more sequence changes compared to a reference polypeptide (e.g., a wild-type polypeptide) or a nucleic acid encoding the reference polypeptide. In some embodiments, a variant polypeptide contains artificially introduced changes in amino acid sequence (e.g., changes in amino acid sequence generated in a laboratory or other facility by human intervention). Such variant polypeptides can contain a single change or multiple changes in amino acid sequence compared to the reference polypeptide. Such amino acid changes include, but are not limited to, substitutions, insertions, and deletions. Guidance for substitutions, insertions, or deletions can be based on alignment of the amino acid sequences of different variant proteins or proteins from different species.

[0027] As used herein, the term "mutant SPINK9 domain" refers to a sequence in a polypeptide that contains one or more amino acid sequence changes compared to wild-type SPINK9 (e.g., SEQ ID NO: 1 or 2). In some embodiments, the mutant SPINK9 domain sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to the wild-type SPINK9 sequence (e.g., SEQ ID NO: 1 or 2). In some embodiments, the sequence changes are artificially introduced sequence changes. Such a "mutant SPINK9 domain" can contain a single change in amino acid sequence or multiple changes in amino acid sequence compared to wild-type SPINK9. For example, in some embodiments, the mutant SPINK9 domain sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sequence differences compared to the wild-type SPINK9 sequence.

[0028] As used herein, the terms "patient," "subject," or "individual" are used interchangeably herein and refer to either a human or a non-human animal. The term non-human animal includes, but is not limited to, mammals such as humans, primates, livestock animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats). In one embodiment, the subject is a human.

[0029] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0030] The term "pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable for or compatible with the treatment of a patient.

[0031] The terms "preventing" or "prevention," when used in connection with a disease (such as Netherton Syndrome) or any other medical condition, are art-recognized and well understood in the art, and include administration of a composition to reduce the frequency of, or delay the onset of, symptoms of the medical condition in, a subject relative to subjects who do not receive the composition. Thus, preventing Netherton Syndrome includes, for example, reducing the number or severity of atopic symptoms of the disease in a population of subjects receiving the prophylactic treatment relative to an untreated control population, and / or delaying the appearance of atopic symptoms of the disease in a population of subjects receiving the prophylactic treatment relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0032] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, to form an active compound (e.g., a compound of the present disclosure) in a host after administration. Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs that use esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference in their entireties. The prodrugs of the present disclosure are metabolized to produce a SPINK9 polypeptide. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0033] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or material useful for formulating a SPINK9 polypeptide or other compounds disclosed herein for pharmaceutical or therapeutic use. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include, as appropriate for the particular dosage form desired, (1) water, any and all solvents, dispersion media, diluents, or other liquid vehicles; (2) dispersing or suspending aids; (3) surfactants and surface active agents; (4) isotonicity agents, thickening agents, or emulsifiers; (5) preservatives, antioxidants, chelating agents, solid binders, lubricants; (6) sugars, such as dextran, lactose, glucose, and sucrose; (7) starches, such as corn starch and potato starch; (8) cellulose, and sodium carboxymethylcellulose, ethyl cellulose, and the like. Examples of suitable cellulose derivatives include cellulose acetate and the like, (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) buffers such as magnesium hydroxide and aluminum hydroxide, isotonic saline, Ringer's solution, and phosphate buffer solution, (14) osmolality adjusters, and (15) stabilizers. (Remington's The Science and Practice of Pharmacy, 21) stEdition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier is incompatible with the substance or its derivatives, for example, by causing any undesirable biological effects or by otherwise interacting in a deleterious manner with any other component(s) of the composition, its use is contemplated within the scope of the present disclosure.

[0034] As used herein, the term "sequence identity" refers to the sequence similarity between two peptide / polypeptide / protein molecules. For example, when a position in both of the two compared sequences is occupied by the same amino acid monomer subunit, the molecules are identical at that position. Without being bound by any particular theory or methodology, the percent sequence identity between two sequences can be considered as a function of the number of identical positions shared by the two sequences, divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are the same, the two sequences have 60% sequence identity. Generally, comparison is performed when the two sequences are aligned to give the maximum number of identical positions.

[0035] As used herein, the term "SPINK9 polypeptide" refers to a polypeptide comprising a mutant SPINK9 domain and, optionally, an additional domain (e.g., an Fc portion). In certain embodiments, a "SPINK9 polypeptide" comprises two or more mutant SPINK9 domains (e.g., 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, or 2 mutant SPINK9 domains) and, optionally, an additional domain (e.g., an Fc portion). In some embodiments, a SPINK9 polypeptide can comprise multiple associated polypeptide chains (e.g., as polypeptide dimers linked through an Fc portion or other dimerization domain). In some embodiments, each polypeptide chain in a SPINK9 polypeptide comprises a mutant SPINK9 domain.

[0036] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required for a subject will depend, for example, on the subject's size, health, and age, as well as the nature and extent of the condition being treated. Those skilled in the art can readily determine the effective amount for a given situation by routine experimentation.

[0037] The terms "treat," "treating," and "treatment," as understood in the art, refer to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, a stable (i.e., not worsening) disease state, preventing the spread of disease, delaying or slowing the progression of disease, improvement or palliation of the disease state, and remission (partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0038] Amino acid residues may be referred to by their corresponding three letter codes or by their corresponding one letter codes. Various three letter and one letter codes for the 20 commonly used amino acids are provided below. TIFF2025526298000001.tif79169

[0039] overview The present disclosure provides SPINK9 polypeptides comprising (e.g., at least one) mutant SPINK9 domain. The SPINK9 polypeptide inhibits or otherwise attenuates active KLK through interaction of the mutant SPINK9 domain with the active KLK. The active KLK can be any member of the KLK family, including KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, or any combination thereof. In some embodiments, the SPINK9 polypeptide inhibits the activity of any one of KLK5, KLK7, KLK14, or any combination thereof. In some embodiments, the SPINK9 polypeptide inhibits the activity of KLK5.

[0040] In some embodiments, the SPINK9 polypeptide is a selective inhibitor of an active KLK (e.g., a selective inhibitor of KLK5). As used herein, the term "selective inhibitor" does not limit inhibition to only the targeted KLK (e.g., KLK5); a selective inhibitor may also inhibit the activity of additional KLK family members or serine proteases, provided that the activity of the target KLK (e.g., KLK5) is inhibited to a greater extent (e.g., by at least 20% or more, such as by 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more) compared to the activity of another KLK family member or other serine protease.

[0041] In a preferred embodiment, the SPINK9 polypeptide inhibits KLK5 and KLK14. In another preferred embodiment, the SPINK9 polypeptide is a selective inhibitor of KLK5 and KLK14.

[0042] In a preferred embodiment, the SPINK9 polypeptide inhibits KLK5. In another preferred embodiment, the SPINK9 polypeptide is a selective inhibitor of KLK5.

[0043] Mutant SPINK9 domain The mutant SPINK9 domain can comprise all or a portion of the wild-type SPINK9 sequence of SEQ ID NO: 1 (including the signal peptide), or all or a portion of the wild-type SPINK9 sequence of SEQ ID NO: 2 (excluding the signal sequence). In a preferred embodiment, the mutant SPINK9 domain comprises all or a portion of the wild-type SPINK9 of SEQ ID NO: 2. MRATAIVLLLALTLATMFSIECAKQTKQMVDCSHYKKLPPGQQRFCHHMYDPICGSDGKTYKNDCFFCSKVKKTDGTLKFVHFGKC (SEQ ID NO: 1) IECAKQTKQMVDCSHYKKLPPGQQRFCHHMYDPICGSDGKTYKNDCFFCSKVKKTDGTLKFVHFGKC (SEQ ID NO: 2)

[0044] In some embodiments, the variant SPINK9 domain, or biologically active fragment thereof, shares 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or 2. In certain embodiments, the variant SPINK9 domain, or biologically active fragment thereof, shares 70% or more (i.e., 75%, 80%, 85%, 90%, or 95% or more) sequence identity to SEQ ID NO: 1 or 2. In preferred embodiments, the variant SPINK9 domain, or biologically active fragment thereof, shares 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In another preferred embodiment, the variant SPINK9 domain, or biologically active fragment thereof, has 70% or more (i.e., 75%, 80%, 85%, 90%, or 95% or more) sequence identity to SEQ ID NO: 2.

[0045] The variant SPINK9 domain can contain one or more amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the variant SPINK9 domain contains 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the variant SPINK9 domain contains 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, or 7 amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the variant SPINK9 domain contains 2 to 15 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid changes relative to SEQ ID NO: 1 or 2. Preferably, the variant SPINK9 domain contains more than 1 and fewer than 20 (eg, 2-7 or 2-15) amino acid changes relative to SEQ ID NO: 1 or 2.

[0046] In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain occurs at or within amino acid positions 40-53 of SEQ ID NO: 1 or positions 21-34 of SEQ ID NO: 2. Preferably, at least one of the one or more amino acid changes in the mutant SPINK9 domain occurs at or within amino acid positions 21-34 of SEQ ID NO: 2. The referenced amino acid positions encompass the reactive P1 / P1' active site of SPINK9.

[0047] In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at or within positions 20-28 of SEQ ID NO: 1, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 40-53 of SEQ ID NO: 1. In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at or within positions 1-9 of SEQ ID NO: 2, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 21-34 of SEQ ID NO: 2. Preferably, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at positions 1-9 of SEQ ID NO: 2, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 21-34 of SEQ ID NO: 2.

[0048] In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at or within positions 20-24 of SEQ ID NO: 1, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 40-53 of SEQ ID NO: 1. In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at or within positions 1-5 of SEQ ID NO: 2, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 21-34 of SEQ ID NO: 2. Preferably, at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution or deletion of one or more consecutive amino acids at or within positions 1-5 of SEQ ID NO: 2, and at least one of the one or more amino acid changes in the mutant SPINK9 domain is a substitution occurring at or within positions 21-34 of SEQ ID NO: 2.

[0049] In some embodiments, at least 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the one or more amino acid changes in the mutant SPINK9 domain occur at or within amino acid positions 40-53 of SEQ ID NO: 1 or positions 21-34 of SEQ ID NO: 2. Preferably, at least 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the one or more amino acid changes in the mutant SPINK9 domain occur at or within amino acid positions 21-34 of SEQ ID NO: 2.

[0050] In one embodiment, the variant SPINK9 domain comprises a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid positions 1-20 and 35-67 of SEQ ID NO: 2, and comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO: 2. In a preferred embodiment, the sequence identity to amino acid positions 1-20 and 35-67 of SEQ ID NO: 2 is at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0051] In one embodiment, the mutant SPINK9 polypeptide comprises at least one mutant SPINK9 domain, wherein the at least one mutant SPINK9 domain comprises an amino acid sequence that shares at least 70% sequence identity with amino acids 1-20 and 35-67 set forth in SEQ ID NO:2, and the mutant SPINK9 domain further comprises at least one amino acid substitution at positions 21-34 of SEQ ID NO:2.

[0052] In one embodiment, the mutant SPINK9 polypeptide comprises, from N-terminus to C-terminus, a first amino acid sequence corresponding to amino acids 1 to 20 of SEQ ID NO:2, a second amino acid sequence corresponding to amino acids 21 to 34 of SEQ ID NO:2, and a third amino acid sequence corresponding to amino acids 35 to 67 of SEQ ID NO:2.

[0053] In a first aspect of such embodiments, (i) a first amino acid sequence shares at least 70% sequence identity with amino acids 1-20 of SEQ ID NO:2; (ii) a second amino acid sequence comprises at least one amino acid substitution with amino acids 21-34 of SEQ ID NO:2; and (iii) a third amino acid sequence shares at least 70% sequence identity with amino acids 35-67 of SEQ ID NO:2, and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0054] In a second aspect of such an embodiment, (i) the first amino acid sequence optionally comprises an amino acid at position 1, 2, 3, 4, 5, 6, 7, 8, or 9 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) to positions 21-34 of SEQ ID NO:2; and (iii) the third amino acid sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2 and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0055] In a third aspect of such embodiments, (i) the first amino acid sequence optionally comprises an amino acid at position 1, 2, or 3 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) the third amino acid sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2 and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0056] In a fourth aspect of such embodiments, (i) the first amino acid sequence comprises a conservative amino acid substitution at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 of SEQ ID NO:2, and optionally shares at least 70% sequence identity to amino acids 10-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) the third amino acid sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2, and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0057] In a fifth aspect of such embodiments, (i) the first amino acid sequence comprises a conservative amino acid substitution at any one of positions 1, 2, or 3 of SEQ ID NO:2, and optionally shares at least 70% sequence identity to amino acids 4-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) the third amino acid sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2, and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0058] In a sixth aspect of such embodiments, (i) the first amino acid sequence comprises a deletion of at least one, two, or three amino acids at positions 1, 2, or 3 of SEQ ID NO:2, and optionally shares at least 70% sequence identity to amino acids 4-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) the third amino acid sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2, and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0059] In a seventh aspect of such embodiments, (i) a first amino acid sequence comprises a deletion of amino acids at positions 1, 2, and 3 of SEQ ID NO:2, and optionally shares at least 70% sequence identity to amino acids 4-20 of SEQ ID NO:2; (ii) a second amino acid sequence comprises at least one amino acid change (e.g., an amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) a sequence shares at least 70% (i.e., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2, and optionally comprises an amino acid change (i.e., an amino acid substitution) at position 43.

[0060] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes (eg, amino acid substitutions) at positions 21 to 34 of SEQ ID NO:2.

[0061] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (eg, amino acid substitutions) at positions 21 to 34 of SEQ ID NO:2.

[0062] In any one of the first to seventh aspects above, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (e.g., amino acid substitutions) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO:2, and optionally one or more amino acid changes (e.g., amino acid substitutions) at positions 21, 23, 27, 31, 32, 33, and 34 of SEQ ID NO:2.

[0063] In any one of the above first to seventh aspects, the second amino acid sequence contains at least one amino acid change (e.g., 1, 2, 3, 4, 5, 6, or 7) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO: 2. For example, the second amino acid sequence can contain amino acid changes at only one position (e.g., position 28), only two positions (e.g., positions 28 and 29), only three positions (e.g., positions 28, 29, and 30), only four positions (e.g., positions 25, 28, 29, and 30), only five positions (e.g., positions 25, 26, 28, 29, and 30), only six positions (e.g., positions 22, 25, 26, 28, 29, and 30), or all seven positions (e.g., positions 22, 24, 25, 26, 28, 29, and 30).

[0064] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (e.g., amino acid substitutions) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO: 2, wherein (i) the amino acid at position 22 is selected from Gly, Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; (ii) the amino acid at position 24 is selected from Gln, Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; or (iii) the amino acid at position 25 is selected from Gly, Glu, Met, Gln, Ala, Leu, Val, Asn, Ser, or Tyr; is selected from Arg, Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; (iv) the amino acid at position 26 is selected from Phe, Tyr, Trp, Met, His, Lys, or Asn; (v) the amino acid at position 28 is selected from His, Thr, Ser, or Gly; (vi) the amino acid at position 29 is selected from His, Arg, or Lys; (vii) the amino acid at position 30 is selected from Met, Glu, or Asp; or (viii) any combination thereof.

[0065] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO:2, wherein the second amino acid sequence (i) comprises a substitution of Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His amino acid for the amino acid corresponding to position 22 of SEQ ID NO:2, or the amino acid at position 22 of SEQ ID NO:2 is Gly, (ii) comprises a substitution of Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr amino acid for the amino acid corresponding to position 24 of SEQ ID NO:2, or the amino acid at position 24 of SEQ ID NO:2 is Gln, and (iii) comprises a substitution of Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or (iv) a substitution of a Tyr, Trp, Met, His, Lys, or Asn amino acid for the amino acid at position 26 of SEQ ID NO:2, or the amino acid at position 26 of SEQ ID NO:2 is Phe; (v) a substitution of a Thr, Ser, or Gly amino acid for the amino acid at position 28 of SEQ ID NO:2, or the amino acid at position 28 of SEQ ID NO:2 is His; (vi) a substitution of an Arg or Lys amino acid for the amino acid at position 29 of SEQ ID NO:2, or the amino acid at position 29 of SEQ ID NO:2 is His; (vii) a substitution of a Glu or Asp amino acid for the amino acid at position 30 of SEQ ID NO:2, or the amino acid at position 30 of SEQ ID NO:2 is Met; or (viii) any combination thereof.

[0066] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (e.g., amino acid substitutions) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO: 2, wherein (i) the amino acid at position 22 is selected from Gly, Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; (ii) the amino acid at position 24 is selected from Gln, Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; (i) the amino acid at position 25 is selected from Arg, Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; (iv) the amino acid at position 26 is selected from Phe, Tyr, Trp, Met, His, Lys, or Asn; (v) the amino acids at positions 28 to 30 are selected from Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, or Gly-Arg-Asp; or (vi) any combination thereof.

[0067] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO:2, and the second amino acid sequence (i) comprises a substitution of Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His amino acid for the amino acid at position 22 of SEQ ID NO:2, or the amino acid at position 22 of SEQ ID NO:2 is Gly, (ii) comprises a substitution of Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr amino acid for the amino acid at position 24 of SEQ ID NO:2, or the amino acid at position 24 of SEQ ID NO:2 is Gln, and (iii) comprises a substitution of Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr amino acid for the amino acid at position 24 of SEQ ID NO:2. (iv) the amino acid at position 26 of SEQ ID NO:2 is substituted with a Tyr, Trp, Met, His, Lys, or Asn amino acid, or the amino acid at position 26 of SEQ ID NO:2 is Phe; (v) the amino acids at positions 28-30 of SEQ ID NO:2 are substituted with a Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, or Gly-Arg-Asp amino acid; or (vi) any combination thereof.

[0068] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (e.g., amino acid substitutions) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO: 2, wherein (i) the amino acid at position 22 is selected from Gly, Glu, or Met; (ii) the amino acid at position 24 is selected from Gln, Glu, or Thr; (iii) the amino acid at position 25 is selected from Leu, Ile, His, Glu, or Trp; (iv) the amino acid at position 26 is selected from Phe, Tyr, or Trp; (v) the amino acid at position 28 is selected from Thr, Ser, or Gly; (vi) the amino acid at position 29 is selected from Arg or Lys; (vii) the amino acid at position 30 is selected from Glu or Asp; or (viii) any combination thereof.

[0069] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO:2, wherein the second amino acid sequence (i) comprises a substitution of a Glu or Met amino acid for the amino acid at position 22 of SEQ ID NO:2, or the amino acid at position 22 of SEQ ID NO:2 is Gly, (ii) comprises a substitution of a Glu or Thr amino acid for the amino acid at position 24 of SEQ ID NO:2, or the amino acid at position 24 of SEQ ID NO:2 is Gln, (iii) comprises a substitution of a Leu, Ile, His, Glu, or Trp amino acid for the amino acid at position 25 of SEQ ID NO:2, or the amino acid at position 25 of SEQ ID NO:2 is A rg, (iv) comprising a substitution of a Tyr or Trp amino acid for the amino acid at position 26 of SEQ ID NO:2, or the amino acid at position 26 of SEQ ID NO:2 is Phe, (v) comprising a substitution of a Thr, Ser, or Gly amino acid for the amino acid at position 28 of SEQ ID NO:2, or the amino acid at position 28 of SEQ ID NO:2 is His, (vi) comprising a substitution of an Arg or Lys amino acid for the amino acid at position 29 of SEQ ID NO:2, or the amino acid at position 29 of SEQ ID NO:2 is His, (vii) comprising a substitution of a Glu or Asp amino acid for the amino acid at position 30 of SEQ ID NO:2, or the amino acid at position 30 of SEQ ID NO:2 is Met, or (viii) any combination thereof.

[0070] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid changes (e.g., amino acid substitutions) at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO: 2, wherein (i) the amino acid at position 22 is selected from Gly, Glu, or Met; (ii) the amino acid at position 24 is selected from Gln, Glu, or Thr; (iii) the amino acid at position 25 is selected from Leu, Ile, His, Glu, or Trp; (iv) the amino acid at position 26 is selected from Phe, Tyr, or Trp; (v) the amino acids at positions 28-30 are selected from Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, or Gly-Arg-Asp; or (vi) any combination thereof.

[0071] In any one of the above first to seventh aspects, the second amino acid sequence comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions at positions 22, 24, 25, 26, 28, 29, and 30 of SEQ ID NO:2, and the second amino acid sequence (i) comprises a substitution of a Glu or Met amino acid for the amino acid at position 22 of SEQ ID NO:2, or the amino acid at position 22 of SEQ ID NO:2 is Gly, (ii) comprises a substitution of a Glu or Thr amino acid for the amino acid at position 24 of SEQ ID NO:2, or the amino acid at position 24 of SEQ ID NO:2 is Gln, and (iii) comprises a substitution of a Glu or Thr amino acid for the amino acid at position 25 of SEQ ID NO:2. (iv) the amino acid at position 26 of SEQ ID NO:2 is substituted with a Tyr or Trp amino acid, or the amino acid at position 26 of SEQ ID NO:2 is Phe; (v) the amino acids at positions 28-30 of SEQ ID NO:2 are substituted with a Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, or Gly-Arg-Asp amino acid; or (vi) any combination thereof.

[0072] In any one of the above first to seventh aspects, the optional amino acid change at position 43 of the third amino acid sequence is an amino acid substitution. In any one of the above first to seventh aspects, the third amino acid sequence comprises a substitution of a Gly amino acid for the amino acid at position 43 of SEQ ID NO:2.

[0073] In any one of the above first to seventh aspects, at least one of the amino acid changes in SEQ ID NO: 2 is an amino acid substitution. In any one of the above first to seventh aspects, the amino acid change in SEQ ID NO: 2 is an amino acid substitution.

[0074] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of the amino acid sequence represented by Formula I (SEQ ID NO: 3), provided that the amino acid sequence represented by Formula I does not correspond to the wild-type sequence of SPINK9 (i.e., SEQ ID NO: 1 or 2). Ile Glu Cys Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro X1Gln X2X3X4Cys X5X6X7Tyr Asp Pro Ile Cys Gly Ser Asp Gly Lys Thr Tyr Cys (Formula I, SEQ ID NO: 3).

[0075] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of the amino acid sequence represented by Formula III (SEQ ID NO: 135), provided that the amino acid sequence represented by Formula III does not correspond to the wild-type sequence of SPINK9 (i.e., SEQ ID NO: 1 or 2). X9X 10 X 11 X 12 X 13 X14 X 15 X 16 X 17 Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro

[0076] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of the amino acid sequence represented by Formula IV (SEQ ID NO: 136), provided that the amino acid sequence represented by Formula IV does not correspond to the wild-type sequence of SPINK9 (i.e., SEQ ID NO: 1 or 2). X9X 10 X 11 Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys Lys Leu Pro Pro X1Gln Cys (Formula I, SEQ ID NO: 136).

[0077] In preferred embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of an amino acid sequence represented by Formula I, Formula III, or Formula IV, wherein X1-X 17 is as shown in Table 1 or 2 herein. [Table 1] [Table 2]

[0078] In another embodiment, the mutant SPINK9 domain comprises, consists of, or consists essentially of an amino acid sequence represented by Formula I, Formula III, or Formula IV, wherein X1-X 17 is as shown in Table 3 or 4 herein. [Table 3] [Table 4]

[0079] In certain preferred embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-23 and 115-134 shown in Table 5. [Table 5-1] [Table 5-2]

[0080] In some embodiments, the variant SPINK9 domain has a sequence disclosed herein (such as SEQ ID NOS:4-23 and 115-134) that further comprises one or more (e.g., 1-20, 1-15, 1-10, 1-8, or 1-5) conservative sequence modifications, wherein the conservative sequence modification(s) are not at amino acid positions 41, 43, 44, 45, 47, 48, or 49 of SEQ ID NO:1 or at amino acid positions 22, 24, 25, 26, 28, 29, or 30 of SEQ ID NO:2. In some embodiments, the variant SPINK9 domain has a sequence disclosed herein further comprising one or more (e.g., 1-20, 1-15, 1-10, 1-8, or 1-5) conservative sequence modifications, wherein the conservative sequence modification(s) are not at amino acid positions 32, 41, 43, 44, 45, 46, 47, 48, 49, 54, 65, 68, or 86 of SEQ ID NO: 1, or 13, 22, 24, 25, 27, 26, 28, 29, 30, 35, 46, 49, or 67 of SEQ ID NO: 2. As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or alter the interaction of the variant SPINK9 domain and a cognate peptide, e.g., a KLK protease, such as KLK5, optionally including natural variations in the amino acid sequence of SPINK9. Such conservative substitutions include, but are not limited to, amino acid substitutions, additions (e.g., adding an amino acid to the N- or C-terminus of the peptide), and deletions (e.g., deleting an amino acid from the N- or C-terminus of the peptide). In one embodiment, a conservative amino acid modification is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid and glutamic acid), neutral side chains (e.g., cysteine, serine, and threonine, asparagine, and glutamine), alkyl / aliphatic side chains (e.g., glycine, alanine, valine, methionine, leucine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, and tryptophan).Thus, in certain embodiments, one or more amino acid residues of the peptides described herein can be replaced with other amino acid residues from the same side chain family, and the altered peptides can be tested for retention of target binding / inhibition using methods known in the art. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0081] SPINK9 polypeptide The present disclosure provides SPINK9 polypeptides comprising (e.g., at least one) mutant SPINK9 domain. In some embodiments, the SPINK9 polypeptide comprises a mutant SPINK9 domain and (e.g., at least one) additional domain. Any SPINK9 polypeptide described herein may be provided as a pharmaceutically acceptable salt. Any mutant SPINK9 domain described herein may be used in a SPINK9 polypeptide.

[0082] Additional domains may be selected to provide or improve properties or characteristics of the SPINK9 polypeptide, including, but not limited to, promoting solubility, promoting storage, increasing in vivo half-life, increasing in vivo stability, increasing storage half-life, increasing storage stability, reducing immunogenicity, reducing toxicity, providing targeting to a particular cell type, providing delayed or controlled release in vivo, or any combination of the foregoing. Exemplary additional domains are discussed herein.

[0083] In some embodiments, the SPINK9 polypeptide comprises a single mutant SPINK9 domain, which is any mutant SPINK9 domain disclosed herein, including, but not limited to, a mutant SPINK9 domain comprising the amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5.

[0084] In some embodiments, the SPINK9 polypeptide comprises 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) independently selected variant SPINK9 domains, each variant SPINK9 domain being any variant SPINK9 domain disclosed herein, including, but not limited to, a variant SPINK9 domain comprising an amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5.

[0085] In some embodiments, the SPINK9 polypeptide comprises first and second independently selected mutant SPINK9 domains, each of which comprises an independently selected amino acid sequence selected from the mutant SPINK9 domain amino acid sequences disclosed herein, including, but not limited to, the amino acid sequences of Formula I, III, or IV and the amino acid sequences set forth in Tables 1-5. In some such embodiments, each of the first and second mutant SPINK9 domains is identical. In some such embodiments, each of the first and second mutant SPINK9 domains is not identical. In preferred embodiments, each mutant SPINK9 domain is independently selected from Table 5.

[0086] In some embodiments, the SPINK9 polypeptide comprises first, second, and third independently selected mutant SPINK9 domains, each of which comprises an independently selected amino acid sequence selected from the mutant SPINK9 domain amino acid sequences disclosed herein, including, but not limited to, the amino acid sequences of Formula I, III, or IV and those set forth in Tables 1-5. In some such embodiments, two and only two of the mutant SPINK9 domains are identical. In some such embodiments, each of the first, second, and third mutant SPINK9 domains is identical. In some such embodiments, each of the first, second, and third mutant SPINK9 domains is not identical. In a preferred embodiment, each mutant SPINK9 domain is independently selected from Table 5.

[0087] When a SPINK9 polypeptide comprises two or more (e.g., 2-20) mutant SPINK9 domains, the mutant SPINK9 domains can be positioned adjacent to one another in the SPINK9 polypeptide, or additional domains can be positioned between one or more mutant SPINK9 domains. In some embodiments, all mutant SPINK9 domains in a SPINK9 polypeptide are positioned adjacent to one another.

[0088] In some embodiments, when two or more adjacent mutant SPINK9 domains are present, the mutant SPINK9 domains are joined by direct fusion (e.g., there are no intervening amino acid sequences disposed between adjacent mutant SPINK9 domains). In some embodiments, when two or more adjacent mutant SPINK9 domains are present, the mutant SPINK9 domains are joined by a linker sequence. In some embodiments, when three or more mutant SPINK9 domains are present, the mutant SPINK9 domains are joined by a combination of direct fusion and linker sequences. In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is joined to an adjacent mutant SPINK9 domain by a linker sequence. In other embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is joined to an adjacent mutant SPINK9 domain by direct fusion.

[0089] In some embodiments, a SPINK9 polypeptide comprises multiple associated polypeptide chains (e.g., polypeptide dimers linked through an Fc portion or other dimerization domain), and at least one polypeptide chain in the SPINK9 polypeptide comprises at least one (e.g., 2-20) mutant SPINK9 domain.

[0090] In some embodiments, a SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain linked through an Fc portion or other dimerization domain, and at least one of the first and second polypeptide chains comprises at least one (e.g., 2-20) mutant SPINK9 domain. In some such embodiments, the mutant SPINK9 domains are independently selected from any mutant SPINK9 domain disclosed herein, including, but not limited to, a mutant SPINK9 domain comprising the amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5. In a preferred embodiment, the first and second mutant SPINK9 domains are independently selected from Table 5.

[0091] In some embodiments, a SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain linked through an Fc portion or other dimerization domain, wherein the first polypeptide chain comprises a first mutant SPINK9 domain and the second polypeptide chain comprises a second mutant SPINK9 domain. In some such embodiments, the mutant SPINK9 domains are independently selected from any mutant SPINK9 domain disclosed herein, including, but not limited to, a mutant SPINK9 domain comprising the amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5. In some such embodiments, each of the first and second mutant SPINK9 domains is identical. In some such embodiments, each of the first and second mutant SPINK9 domains is not identical. In a preferred embodiment, the first and second mutant SPINK9 domains are independently selected from Table 5.

[0092] In some embodiments, a SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain linked through an Fc portion or other dimerization domain, wherein the first polypeptide chain comprises a first and a second mutant SPINK9 domain, and the second polypeptide chain comprises a third and a fourth mutant SPINK9 domain. In some such embodiments, the mutant SPINK9 domains are independently selected from any mutant SPINK9 domain disclosed herein, including, but not limited to, a mutant SPINK9 domain comprising the amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5. In some such embodiments, at least two of the first through fourth mutant SPINK9 domains are identical. In some such embodiments, each of the first through fourth mutant SPINK9 domains is identical. In some such embodiments, each of the first through fourth mutant SPINK9 domains is not identical. In preferred embodiments, the first through fourth mutant SPINK9 domains are independently selected from Table 5.

[0093] In some embodiments, a SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain linked through an Fc portion or other dimerization domain, wherein the first polypeptide chain comprises first, second, and third mutant SPINK9 domains, and the second polypeptide chain comprises fourth, fifth, and sixth mutant SPINK9 domains. In some such embodiments, the mutant SPINK9 domains are independently selected from any mutant SPINK9 domain disclosed herein, including, but not limited to, a mutant SPINK9 domain comprising the amino acid sequence of Formula I, III, or IV, or an amino acid sequence set forth in Tables 1-5. In some such embodiments, at least two of the first through sixth mutant SPINK9 domains are identical. In some such embodiments, each of the first through sixth mutant SPINK9 domains is identical. In some such embodiments, each of the first through sixth mutant SPINK9 domains is not identical. In preferred embodiments, the first through sixth mutant SPINK9 domains are independently selected from Table 5.

[0094] When a SPINK9 polypeptide comprises first and second polypeptide chains, and the first and / or second polypeptide chains comprise two or more (e.g., 2-20) mutant SPINK9 domains, the mutant SPINK9 domains in the first and / or second polypeptide chains can be positioned adjacent to one another in the first and / or second polypeptide chains, or additional domains can be positioned between one or more mutant SPINK9 domains in the first and / or second polypeptide chains. In some embodiments, all of the mutant SPINK9 domains in the first and / or second polypeptide chains are positioned adjacent to one another.

[0095] In some embodiments, when two or more adjacent mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are joined by direct fusion (e.g., there are no intervening amino acid sequences disposed between adjacent mutant SPINK9 domains). In some embodiments, when two or more adjacent mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are joined by a linker sequence. In some embodiments, when three or more mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are joined by a combination of direct fusion and linker sequences. In some embodiments, at least one mutant SPINK9 domain on the first and / or second polypeptide chains is joined to an adjacent mutant SPINK9 domain by a linker sequence. In other embodiments, at least one mutant SPINK9 domain on the first and / or second polypeptide chains is joined to an adjacent mutant SPINK9 domain by direct fusion.

[0096] SPINK9 polypeptides may further comprise additional domains. In some embodiments, the SPINK9 polypeptide comprises one to five additional domains. Preferably, the additional domain(s) provide the SPINK9 polypeptide with additional functions in addition to inhibiting active kallikrein-related peptidase (e.g., KLK5).

[0097] In a preferred embodiment, the additional domain is an immunoglobulin fragment crystallizable (Fc) region, or a functional fragment thereof. The Fc region has been shown to bind to the neonatal Fc receptor (FcRn) on endothelial cells lining blood vessels; upon binding, the Fc region containing the polypeptide is protected from degradation and re-released into the circulation, thereby increasing the half-life of the bound polypeptide after administration to a subject. The Fc region may be attached to a SPINK9 polypeptide to optimize the pharmacokinetic and pharmacodynamic properties of the polypeptide.

[0098] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region can have the native amino acid sequence or can contain one or more modifications. The Fc region of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. Changes in the amino acid sequence of the Fc portion that alter antibody effector functions are known in the art (e.g., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc region mediates several important effector functions, including, but not limited to, cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), as well as increasing the half-life and decreasing the clearance rate of the polypeptide. In some cases, these effector functions are desirable for therapeutic immunoglobulins, but in other cases, they may be unnecessary or even deleterious, depending on the therapeutic purpose.

[0099] In some embodiments, the Fc region, or functional fragment thereof, is attached to the SPINK9 polypeptide by direct fusion or by a linker sequence, hi some embodiments, the Fc region, or functional fragment thereof, may be attached to the SPINK9 polypeptide at the N-terminus or C-terminus of the mutant SPINK9 domain.

[0100] In some embodiments, the immunoglobulin Fc region is derived from a human. In some embodiments, the immunoglobulin Fc region is derived from a non-human animal, including, but not limited to, a rodent, such as, but not limited to, a mouse and a rat; a mammal, such as, but not limited to, a rabbit, a cow, a pig, a dog, a cynomolgus monkey, a marmoset, or a rhesus monkey; or an avian, such as, but not limited to, a chicken. Preferably, the immunoglobulin Fc region is derived from a human or a non-human animal and is "humanized," as known in the art. In some embodiments, the immunoglobulin Fc region comprises a human Fc sequence selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM Fc regions.

[0101] In some embodiments, the SPINK9 polypeptide comprises a modified Fc region. In some embodiments, the SPINK9 polypeptide comprises a modified Fc region of human origin. The modified Fc region can include amino acid insertions, deletions, substitutions, or chemical modifications. For example, the Fc region can be modified to increase or decrease complement binding, increase or decrease ADCC or CDC, increase or decrease FcR binding, increase or decrease FcRn binding, modify glycosylation, or any combination thereof. Various Fc modifications are known in the art and are described, for example, in Labrijin et al. (2009) Nature Biotech. 27(8):767-771, Greenwood et al. (1993) Eur. J. Immunol. 23:1098-1104, Mueller et al. (1997) Mol. Immunol. 34:441-452, Rother et al. (2007) Nature Biotechnol. 25:1256-1264, Lee et al. (2019) Nature Comm. 10, article number 5031, and Saunders, Front. Immunol. (2019) article 1296. Any of the Fc modifications known in the art can be applied to SPINK9 polypeptides comprising the Fc region disclosed herein. In some embodiments, the Fc modification is an amino acid substitution.

[0102] In some embodiments, SPINK9 polypeptides comprising modified Fc regions are characterized by reduced binding (e.g., minimal or absent binding) to human Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or a combination thereof). In some embodiments, SPINK9 polypeptides comprising modified Fc regions exhibit at least a 5% or greater (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) reduction in Fc receptor binding compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, SPINK9 polypeptides comprising modified Fc regions exhibit at least a 5% or greater (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) reduction in ADCC compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, reduced ADCC is achieved by amino acid substitutions L234A / L235A, G237A, N297A, or any combination thereof (numbering according to EU numbering, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969).

[0103] In some embodiments, SPINK9 polypeptides comprising a modified Fc region are characterized by reduced binding (e.g., minimal or no binding) to complement proteins compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, SPINK9 polypeptides comprising a modified Fc region exhibit at least a 5% or greater (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) reduction in C1q binding compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, SPINK9 polypeptides comprising a modified Fc region exhibit at least a 5% or greater (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) reduction in CDC compared to SPINK9 polypeptides comprising a wild-type Fc region.

[0104] In some embodiments, SPINK9 polypeptides comprising modified Fc regions are characterized by increased affinity for the human neonatal Fc receptor (FcRn) at low pH (e.g., pH 5.8-6.0) compared to SPINK9 polypeptides comprising wild-type Fc regions. In some embodiments, SPINK9 polypeptides comprising modified Fc regions exhibit at least 5% or greater (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) affinity for FcRn at low pH compared to SPINK9 polypeptides comprising wild-type Fc regions. In some embodiments, increased affinity for the human neonatal Fc receptor (FcRn) at low pH is achieved with the amino acid substitutions M252Y / S254T / T256E, M428L / N434S, H433K / N434F, V264E / L309D / Q311H, V264E / L309D / Q311H / N434S, or L309D / Q311H / N434S (numbering according to EU numbering, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969).

[0105] In some embodiments, SPINK9 polypeptides comprising a modified Fc region exhibit minimal or reduced glycosylation compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, SPINK9 polypeptides comprising a modified Fc region exhibit at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduction in glycosylation compared to SPINK9 polypeptides comprising a wild-type Fc region. In some embodiments, minimal or reduced glycosylation is achieved with the amino acid substitution N297X, where X is any amino acid other than N (e.g., an N297A substitution) (numbering according to EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969)).

[0106] In some embodiments, the Fc region is derived from an IgG1 antibody, particularly a human IgG1 antibody. In some embodiments, the modified Fc region is derived from an IgG1 antibody, particularly a human IgG1 antibody, and comprises one or more amino acid substitutions selected from the group consisting of: L234A / L235A, M252Y / S254T / T256E, M428L / N434S, H433K / N434F, V264E / L309D / Q311H, V264E / L309D / Q311H / N434S, L309D / Q311H / N434S, N297X, where X is any amino acid other than N (e.g., N297A), P228S A330S, P331S, G237A, E233P / L234V / L235A, A327G / A330S / P331S, or L234F / L235E / P331S (numbering according to EU numbering (Edelman, G. Met.) al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969). In some embodiments, one or more additional mutations are included in such an IgG1-modified Fc region. In some embodiments, the human IgG1-modified Fc region has up to 30 (e.g., 25, 20, 15, 10, 9, 8, 7, 6, 5, or 4) mutations compared to the wild-type human IgG1 Fc region sequence or a fragment thereof.

[0107] In some embodiments, the Fc region comprises, consists of, or consists essentially of the amino acid sequence of Formula II (SEQ ID NO: 24): Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro X1Cys Pro Ala Pro X2X3X4Gly X5Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu X6Ile X7Arg X8Pro Glu Val Thr Cys Val Val X9Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr X 10 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val X 11 His X 12 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys X 13 Leu Pro X 14 X 15 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val X 16 His Glu Ala Leu X 17 X 18 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys、 During the ceremony, X1 is P or S, X2 is E or P, X3 and X4 each independently represent L or A, The X5 is G or A, The X6 is M or Y, The X7 is S or T, The X8 is T or E, X9 is V or E, X 10 teeth, N or A, X 11 teeth, L or D, X 12 teeth, Q or H, X 13 teeth, A or G, X 14 teeth, A or S, X 15 teeth, P or S, X 16 teeth, M or L, X 17 teeth, H or K, X 18 teeth, N , S, or F, where wild-type amino acids are underlined and non-underlined amino acids are non-wild-type (substituted) amino acids.

[0108] In some embodiments of Formula II, X6-X7-X8 is MST, and X1-X5 and X9-X 18 In some embodiments of Formula II, X6-X7-X8 is YTE, and X1-X5 and X9-X 18In some embodiments of Formula II, X6-X7-X8 is YTE, and X1-X5 and X9-X 18 Each of the is a wild-type amino acid.

[0109] In some embodiments of Formula II, X 16 and X 18 are M and N, and X1 to X 15 and X 17 In some embodiments of Formula II, at least one of X is a non-wild-type amino acid. 16 and X 18 are L and S, and X1 to X 15 and X 17 In some embodiments of Formula II, at least one of X is a wild-type amino acid. 16 and X 18 are L and S, and X1 to X 15 and X 17 are wild-type amino acids.

[0110] In some embodiments of Formula II, X3-X4 is LL, and X1-X2 and X5-X 18 In some embodiments of Formula II, X3-X4 are AA, and X1-X2 and X5-X6 are non-wild-type amino acids. 18 In some embodiments of Formula II, X3-X4 are AA, and X1-X2 and X5-X6 are AA. 18 are wild-type amino acids.

[0111] In some embodiments of Formula II, X, X 11 , and X 12 are V, L, and Q, and X1 to X8, X 10 , and X 13 ~X 18 In some embodiments of Formula II, at least one of X, X is a non-wild-type amino acid. 11 , and X 12are E, D, and H, and X1 to X8, X 10 , and X 13 ~X 18 In some embodiments of Formula II, at least one of X, X is a wild-type amino acid. 11 , and X 12 are E, D, and H, and X1 to X8, X 10 , and X 13 ~X 18 are wild-type amino acids.

[0112] In some embodiments of Formula II, X 11 , X 12 , and X 18 are L, Q, and N, and X1 to X 10 and X 13 ~X 17 In some embodiments of Formula II, at least one of X is a non-wild-type amino acid. 11 , X 12 , and X 18 are D, H, and S, and X1 to X 10 and X 13 ~X 17 In some embodiments of Formula II, at least one of X is a wild-type amino acid. 11 , X 12 , and X 18 are D, H, and S, and X1 to X 10 and X 13 ~X 17 are wild-type amino acids.

[0113] In some embodiments of Formula II, X 17 -X 18 is HN, and X1~X 16 In some embodiments of Formula II, at least one of X is a non-wild-type amino acid. 17 -X 18 is KF, and X1~X 16 In some embodiments of Formula II, at least one of X is a wild-type amino acid. 17 -X 18 is KF, and X1~X16 are wild-type amino acids.

[0114] In some embodiments of Formula II, X-X is AA and X 16 and X 18 are L and S, and X1 to X2, X5 to X 15 , and X 17 In some embodiments of Formula II, X3-X4 are AA, X6-X7-X8 are YTE, and X1-X2, X5, and X9-X 18 are wild-type amino acids.

[0115] In some embodiments of Formula II, X 10 is N, and X1 to X9 and X 11 ~X 18 In some embodiments of Formula II, at least one of X is a non-wild-type amino acid. 10 is A, and X1 to X9 and X 11 ~X 18 In some embodiments of Formula II, at least one of X is a wild-type amino acid. 10 is A, and X1 to X9 and X 11 ~X 18 are wild-type amino acids.

[0116] In some embodiments of Formula II, X 10 is A, X3-X4 is AA, X1 to X2, X5 to X9, and X 11 ~X 18 At least one or all of are wild-type amino acids.

[0117] In some embodiments of Formula II, X 10 is A and X 16 and X 18 are L and S, X1 to X9, X 11 ~X 15 , and X 17 At least one or all of are wild-type amino acids.

[0118] In some embodiments of Formula II, X 10 is A, X6-X7-X8 are YTE, and X1 to X5, X9, and X 11 ~X 18 At least one or all of are wild-type amino acids.

[0119] In some embodiments of Formula II, X 10 is A, X3-X4 is AA, and X 16 and X 18 are L and S, X1 to X2, X5 to X9, X 11 ~X 15 , and X 17 At least one or all of are wild-type amino acids.

[0120] In some embodiments of Formula II, X 10 is A, X3-X4 is AA, X6-X7-X8 is YTE, and X1 to X2, X5, and X9 to X 18 At least one or all of are wild-type amino acids.

[0121] As used in this disclosure, "linker" or "linker sequence" refers to an amino acid sequence of natural and / or synthetic origin. A linker sequence can comprise a linear amino acid chain in which 20 naturally occurring amino acids are the monomer building blocks. A linker can have a length of 1 to 50 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-30, 30-50 amino acids) and can comprise a repetitive amino acid sequence or a sequence of a naturally occurring polypeptide. In some embodiments, a linker functions to ensure that polypeptides or polypeptide domains within the linked polypeptides are able to perform their biological activity by allowing the polypeptides or polypeptide domains to fold correctly and / or be properly presented. A linker can also function to provide a cleavage site between a mutant SPINK9 domain and / or a mutant SPINK9 domain and an additional domain (e.g., an Fc region). In some embodiments, a linker sequence is rich in glycine, glutamine, and / or serine residues. These residues may be arranged in small repeating units and may be repeated as needed. In some embodiments, the linker comprises a single amino acid, repeated 10-20 times. In some embodiments, the linker comprises a cleavage site, optionally located adjacent to the mutant SPINK9 domain (suitable cleavage sites are known in the art and described herein). For example, but not limited to, the SPINK9 polypeptides described herein can include any linker sequence known in the art, such as a Gly-Gly-Ser repeat linker, including GS, GGS, GGGS (SEQ ID NO:25), GGGSGGGS (SEQ ID NO:26), GGGSGGGSGGGS (SEQ ID NO:27), GGGSGGGSGGGSGGGS (SEQ ID NO:28), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO:29), GGGGS (SEQ ID NO:30), GGGGSGGGGS (SEQ ID NO:31), GGGGSGGGGSGGGGGS (SEQ ID NO:32), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:33), and GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:34).

[0122] In some embodiments, the linker sequence is encoded by a nucleic acid molecule and therefore capable of recombinant expression. In some embodiments, the linker sequence includes one or more bonds that are not peptide bonds (such alternative bonds are known in the art and described herein). In such embodiments, SPINK9 polypeptides containing such linker sequences can be produced by non-recombinant methods described herein, or by a combination of recombinant and non-recombinant methods.

[0123] In some embodiments, the linker sequence comprises a cleavage site. Such a cleavage site can be recognized by a protease naturally occurring in a subject, such as a proprotein convertase (PPC), more preferably a subtilisin-like proprotein convertase (SPC). SPCs are a family of calcium-dependent cleavage enzymes that act on dibasic sites in various peptide / protein substrates. An exemplary SPC is furin. Representative cleavage sites include, but are not limited to, KR↓, RR↓, RX(K / R)R↓, RXXR↓, KXXR↓, RX(V / L)(K / F / L)↓, RNKR↓ (SEQ ID NO: 35), KAKR↓ (SEQ ID NO: 36), VFAQ↓SIP (SEQ ID NO: 37), and RXXRXX(R / K)R↓ (SEQ ID NO: 38), where ↓ indicates the cleavage point. The cleavage sites disclosed herein can be located at the N-terminus, C-terminus, or internal position of any linker sequence disclosed herein. In some embodiments, the cleavage site is located at the N-terminus or C-terminus of any linker sequence disclosed herein. In some embodiments, the cleavage site alone functions as a linker sequence.

[0124] In some embodiments, the SPINK9 polypeptides provided herein further comprise a signal sequence. In some such embodiments, the signal sequence is attached to the amino (N)-terminus of the polypeptide by direct fusion or by a linker sequence. In other embodiments, the signal sequence may be attached to the carboxy (C)-terminus of the polypeptide by direct fusion or by a linker sequence.

[0125] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:39, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:39. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39).

[0126] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:40, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:40. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40).

[0127] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:41, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:41. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 41).

[0128] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:42, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:42. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42).

[0129] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:43, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 43).

[0130] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:44, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44).

[0131] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:45, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:45. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK (SEQ ID NO: 45).

[0132] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:46, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK (SEQ ID NO: 46).

[0133] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:47, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:47. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVEDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47).

[0134] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:48, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVEDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48).

[0135] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:49, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK (SEQ ID NO: 49).

[0136] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:50, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK (SEQ ID NO: 50).

[0137] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:51, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:51. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51).

[0138] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:52, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:52. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 52).

[0139] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:53, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:53. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53).

[0140] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:54, or a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:54. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 54).

[0141] In some preferred embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in Table 6. [Table 6-1] [Table 6-2]

[0142] In some embodiments, the SPINK9 polypeptides described by Table 6 have zero additional mutant SPINK9 domains, and a linker sequence described herein is optionally present to join one or more of the described domains. In certain embodiments, all domains present are joined by direct fusion.

[0143] In some embodiments, the SPINK9 polypeptides described by Table 6 have 1 to 20 (e.g., 1, 2, 3, 4, or 5, 5 to 10, 10 to 15, or 15 to 20) additional variant SPINK9 domains, and a linker sequence described herein is optionally present to join one or more of the listed domains. In certain embodiments, all domains present are joined by direct fusion.

[0144] In some embodiments, the SPINK9 polypeptides described by Table 6 have two additional mutant SPINK9 domains, and a linker sequence described herein is optionally present to join one or more of the described domains. In certain embodiments, all domains present are joined by direct fusion.

[0145] In some embodiments, at least one of the mutant SPINK9 domains is linked to an additional domain of the SPINK9 polypeptide (e.g., the Fc region) by a linker sequence, hi some embodiments, at least one of the mutant SPINK9 domains is linked to an additional domain of the SPINK9 polypeptide (e.g., the Fc region) by direct fusion.

[0146] In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is linked to an adjacent mutant SPINK9 domain by a linker sequence, and the mutant SPINK9 domain is linked to an additional domain (e.g., an Fc region) by a linker sequence or by direct fusion. In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is linked to an adjacent mutant SPINK9 domain by direct fusion, and the mutant SPINK9 domain is linked to an additional domain (e.g., an Fc region) by a linker sequence or by direct fusion.

[0147] In some preferred embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114 as shown in Table 7 below. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9]

[0148] In certain embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:55-114 shown in Table 7 above, wherein 1 to 9 consecutive amino acids are deleted from the N-terminus of the mutant SPINK9 domain (e.g., 1 to 9 consecutive amino acids selected from IECAKQTKQ).

[0149] In certain embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114 shown in Table 7 above, wherein 1 to 3 contiguous amino acids are deleted from the N-terminus of the mutant SPINK9 domain (e.g., 1 to 3 amino acids selected from IEC).

[0150] In certain embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114 shown in Table 7 above, wherein the first three amino acids are deleted from the N-terminus of the mutant SPINK9 domain (e.g., amino acids IEC).

[0151] In some preferred embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 137-139 shown in Table 8 below. [Table 8]

[0152] SPINK9 polypeptides may also include additional modifications that affect the properties of the SPINK9 polypeptide, including, but not limited to, increasing desirable therapeutic properties (e.g., serum half-life) and providing simple means of purification.

[0153] In some embodiments, SPINK9 polypeptides contain one or more bonds other than peptide bonds, such that at least two adjacent amino acids are linked via a bond other than an amide bond. The use of non-peptide bonds can be used to reduce or eliminate undesired proteolysis or degradation, to increase stability in a physiological environment after administration to a subject, to increase or decrease conformational flexibility, or a combination of the foregoing. Suitable non-peptide bonds include, but are not limited to, -CHNH-, -CHS-, -CHCH-, -CH=CH- (cis and trans), -COCH-, -CH(OH)CH-, and -CHSO-. Other alternatives include amide bioisosteres, such as, but not limited to, 1,2,3-triazoles, oxadiazoles, imidazoles, tetrazoles, pyrazoles, indoles, pyridines, pyrazines, inverted and reverse amides, ureas, olefins, fluoroalkenes, trifluoroethylamines, amidines, esters, sulfonamides, phosphonamidates, thioamides, and carbamates (Kumari, (2020) J. Med. Chem. 63(21), 12290-12358).

[0154] In some embodiments, SPINK9 polypeptides are modified by the addition of polyethylene glycol (PEG), PEG mimetics, polypropylene glycol, or polyoxyalkylenes. Such modifications have been shown to increase physical and thermal stability, protect against enzymatic degradation, increase solubility, increase in vivo circulatory half-life, decrease clearance, reduce immunogenicity and antigenicity, and reduce toxicity. Suitable PEG molecules are generally water-soluble at room temperature and have the general formula R(O-CH-CH). n The PEG has the formula OR, where R is hydrogen or a protecting group (e.g., an alkyl or alkanol group), and n is an integer between 1 and 1000. Suitable PEGs can be linear or branched. In some embodiments, suitable PEGs have a number average molecular weight (M) between 4,000 and 10,000 daltons. n ) but other M n can be used. PEG can be attached to the SPINK9 polypeptide via a spacer that includes a terminal reactive group. The spacer can be, for example, a terminal reactive group that mediates a bond between the free amino or carboxyl groups of one or more amino acids and polyethylene glycol.

[0155] In some embodiments, SPINK9 polypeptides are modified by altering the naturally occurring glycosylation pattern. Glycosylation can dramatically affect the physical properties of proteins and may also be important in protein stability, secretion, and subcellular localization. Addition of glycosylation sites can be achieved by altering the amino acid sequence, for example, by substituting one or more naturally occurring amino acids with serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Carbohydrate removal can be achieved chemically or enzymatically, or by substituting codons encoding amino acid residues that are or can be glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exo-glycosidases.

[0156] nucleic acid Nucleic acid molecules encoding the polypeptides described herein are provided herein. The nucleic acids may be present, for example, in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid molecules described herein can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, nucleic acids corresponding to the nucleotide sequences encoding one or more of the polypeptides disclosed herein can be prepared by standard techniques known in the art.

[0157] The present disclosure provides vectors (e.g., viral vectors such as adenovirus-based expression vectors) comprising the nucleic acid molecules described herein. Viral vectors may contain additional DNA segments that can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication or episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Certain vectors are also capable of directing the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, provided herein is an operable nucleic acid linked to one or more regulatory sequences (e.g., promoters) in an expression vector. In some embodiments, the cell transcribes the nucleic acid provided herein, thereby expressing an antibody, antigen-binding fragment thereof, or peptide described herein. The nucleic acid molecule can be integrated into the genome of the cell or can be extrachromosomal.

[0158] In some embodiments, the nucleic acid vectors or recombinant adenoviruses provided herein encode a polypeptide disclosed or otherwise contemplated herein. In some embodiments, the nucleic acid vector comprises a nucleic acid sequence that has undergone codon optimization. In such embodiments, the coding sequence is constructed by varying codons in each nucleic acid used to assemble the coding sequence. Generally, a method for identifying a nucleotide sequence that optimizes codon usage for peptide production comprises at least the following steps (a) through (e): In step (a), oligomers encoding portions of a polypeptide containing degenerate forms of codons for the amino acids encoded in the portions are provided, and the oligomers are extended to provide contiguous coding sequences with overlapping sequences. In step (b), the oligomers are processed to assemble a coding sequence for the peptide. The reassembled peptide is included in an expression system operably linked to control sequences to affect its expression. In step (c), the expression system is transfected into a culture of compatible host cells. In step (d), colonies obtained from the transformed host cells are tested for the level of polypeptide production. In step (e), at least one colony with the highest or satisfactory production of the polypeptide is obtained from the expression system. The portion of the expression system that encodes the protein is sequenced. Further description of codon optimization is provided in U.S. Patent Publication No. 2010 / 035768, which is incorporated by reference for such teachings.

[0159] Also provided herein are host cells containing the nucleic acids or vectors described herein. The polypeptides provided herein (e.g., polypeptides containing at least one mutant SPINK9 domain) can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, can be produced by recombinant DNA techniques, and / or can be chemically synthesized using standard peptide synthesis techniques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding the peptide(s) of the invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous peptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are incorporated herein by reference. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken IM (1981) CRC Crit. Rev. Biochem. 11:255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342, Kent, SBH (1988) Annu. Rev. Biochem. 57:957, and Offord, RE (1980) Semisynthetic Proteins, Wiley Publishing).

[0160] How to use The present invention provides SPINK9 polypeptides, and pharmaceutically acceptable salts thereof, that are useful for treating or preventing diseases or conditions characterized by abnormal kallikrein activity.

[0161] In certain aspects, the present invention provides a SPINK9 polypeptide of the present invention, or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.

[0162] In certain aspects, methods for treating or preventing a disease or condition characterized by abnormal kallikrein activity are provided. The methods include administering an amount of a SPINK9 polypeptide, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, thereby treating or preventing the disease or condition characterized by abnormal kallikrein activity. By reducing kallikrein activity in the subject, the disease or condition characterized by abnormal kallikrein activity is treated. In some embodiments, a therapeutically effective amount is administered to the subject.

[0163] Alternatively, in certain embodiments, the SPINK9 polypeptides disclosed herein, or pharmaceutically acceptable salts thereof, are provided for the treatment of diseases or conditions characterized by aberrant kallikrein activity.

[0164] Alternatively, in certain aspects, the present invention provides the use of a SPINK9 polypeptide disclosed herein, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a disease or condition characterized by abnormal kallikrein activity.

[0165] As used herein, a "disease or condition characterized by abnormal kallikrein activity" refers to any disease or condition in which it is desirable to reduce kallikrein activity (e.g., KLK5 activity). For example, it may be desirable to reduce kallikrein activity in situations of inappropriate or over-activation of kallikrein.

[0166] In certain embodiments, the disease or condition characterized by aberrant kallikrein activity is characterized by aberrant KLK5 activity.

[0167] In certain embodiments, the disease or condition characterized by abnormal kallikrein activity is a skin disease.

[0168] In certain embodiments, the skin disease is eczema, atopic eczema, atopic dermatitis, autosomal recessive ichthyosis, ichthyosiform erythroderma (IE), psoriasis, ultraviolet light-induced skin damage, rosacea, or a skin infection.

[0169] In certain embodiments, the disease or condition is Netherton syndrome.

[0170] In certain embodiments, the disease or condition characterized by abnormal kallikrein activity is selected from the group consisting of hypersensitivity of the immune system (atopy), hyper-IgE syndrome, allergies (including allergies to food and airborne substances), asthma, allergic asthma, chronic inflammation, rhinitis, conjunctivitis, angioedema, eosinophilia, eosinophilic esophagitis, growth retardation, growth failure, trichorrhexis invaginata (TI), bacterial infections of the skin, respiratory tract infections, systemic infections, and gastrointestinal disorders.

[0171] In certain embodiments, the disease or condition is cancer, which may be selected from ovarian cancer, uterine cancer, colorectal cancer, bladder urothelial cancer, oral squamous cell carcinoma, breast cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, head and neck cancer glioma, glioblastoma multiforme, and neuroblastoma.

[0172] composition Disclosed herein are compositions, including pharmaceutical compositions, comprising a SPINK9 polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a cell described herein. The compositions of the invention are useful for treating or preventing various diseases characterized by abnormal kallikrein activity (e.g., abnormal activity of proteases of the kallikrein-related peptidase family, particularly KLK5 activity).

[0173] Pharmaceutical compositions provided herein can contain an amount of a SPINK9 polypeptide, including a therapeutically effective amount, to treat an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human animal. When administered to a human or non-human animal, the composition (or otherwise polypeptide) is preferably administered as a pharmaceutical composition, e.g., comprising a SPINK9 polypeptide disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and their use is contemplated within the scope of the present disclosure, except insofar as any conventional carrier is incompatible with the substance or its derivatives, e.g., by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the composition. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for use via invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion across epithelial barriers), the pharmaceutical composition is pyrogen-free or substantially pyrogen-free. A carrier can be selected, for example, to provide delayed release of the drug, to create a desired pH range, to stabilize the Spink9 polypeptide, to modulate absorption of the Spink9 polypeptide, to modulate desired pharmacokinetic or pharmacodynamic properties of the Spink9 polypeptide, to selectively target one or more cells, tissues, or organs, or any combination of the foregoing.

[0174] Such pharmaceutically acceptable carriers include, but are not limited to, water, other liquid vehicles, solvents, diluents, dispersion media, dispersing or suspending aids, surfactants, surface active agents, isotonic agents, thickening agents, emulsifiers, preservatives, antioxidants (such as ascorbic acid or glutathione), chelating agents, solid binders, lubricants, sugars (such as dextran, lactose, glucose, and sucrose), starches (such as corn starch and potato starch), cellulose, and derivatives thereof (such as sodium carboxymethylcellulose, ethylene glycol, PEG-10 cellulose ... Examples of suitable pharmaceutically acceptable carriers include cellulose acetate, cellulose acetate, and cellulose acetate), oils (peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (propylene glycol, etc.), polyols (glycerin, sorbitol, mannitol, and polyethylene glycol, etc.), esters (ethyl oleate and ethyl laurate, etc.), buffers (magnesium hydroxide and aluminum hydroxide, isotonic saline, Ringer's solution, phosphate buffer solution, etc.), osmolality adjusters, and stabilizers. The choice of pharmaceutically acceptable carrier may depend, for example, on the route of administration of the composition. The pharmaceutical composition preparation may be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, into which, for example, the SPINK9 polypeptide disclosed herein may be incorporated. Liposomes, for example, comprising phospholipids or other lipids, are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0175] Pharmaceutical compositions can be provided in unit dosage forms. The phrase "unit dosage form" refers to physically discrete units, each containing a composition comprising a predetermined amount of Spink9 polypeptide, either alone or in combination with one or more additional therapeutic agents, sufficient to produce the desired effect. It will be understood that the parameters of the unit dosage form depend on the mode of administration, the specific Spink9 polypeptide, and the effect to be achieved. Suitable doses of Spink9 polypeptide are disclosed herein. Pharmaceutical compositions can also be solutions, particularly liquid solutions, suitable for parenteral administration, particularly subcutaneous or intravenous administration. Pharmaceutical compositions can also be in lyophilized or powder form for reconstitution with a liquid solution suitable for parenteral administration, particularly subcutaneous or intravenous administration.

[0176] Pharmaceutical compositions (preparations) can be administered to a subject by any of several routes of administration, including, for example, parenteral administration, oral administration (e.g., as a component of liposomes), administration through the oral mucosa (e.g., sublingually), transdermal administration (e.g., as a patch applied to the skin), topical administration (e.g., as a cream, ointment, or spray applied to the skin), or administration by inhalation. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, subcutaneous, intravenous, intraocular (intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the mode of administration is via subcutaneous administration. In another embodiment, the mode of administration is via intravenous administration.

[0177] Details of suitable routes of administration and compositions suitable therefor can be found, for example, in US Pat. No. 11,246,906 and WO 2022 / 099963.

[0178] The amount of SPINK9 polypeptide that can be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending on the host being treated, the particular mode of administration, and the disease or condition being treated. In some embodiments, the SPINK9 polypeptide is present in a range of about 1 percent to about 99 percent, about 1 percent to about 90 percent, about 1 percent to about 75 percent, about 1 percent to about 50 percent, about 1 percent to about 40 percent, about 1 percent to about 30 percent, about 1 percent to about 20 percent, or about 1 percent to about 10 percent.

[0179] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0180] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0181] Transdermal patches have the additional advantage of providing controlled delivery of the compound of the present invention to the body.Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium.Absorption enhancers can also be used to increase the flux of the compound across the skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0182] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0183] Pharmaceutical compositions suitable for parenteral administration comprise Spink9 polypeptides in combination with one or more pharmaceutically acceptable carriers, such as, but not limited to, sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, antifungals, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0184] These compositions may also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by the inclusion of various antibacterial agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0185] In some cases, it is desirable to slow the absorption of a drug after administration, e.g., subcutaneous administration, in order to prolong the effect of the drug. This can be achieved through the use of depot injections, which release the Spink9 polypeptides disclosed herein over a defined period of time. Depot injections are usually either solid or oil-based and generally contain at least one of the pharmaceutically acceptable carriers described herein. In certain embodiments, injectable depot forms are made by forming microencapsulated matrices of the subject compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable forms can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues. Those skilled in the art are familiar with the possible formulations and uses of depot injections.

[0186] The method of administration may also be provided by an implantable or biodegradable device.

[0187] After the pharmaceutical composition is formulated, it can be stored in a sterile container as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.Such formulations can be stored in a ready-to-use form, a lyophilized form that requires reconstitution before use, a liquid form that requires dilution before use, or other acceptable form.Preferably, the pharmaceutical composition is provided in a disposable container.The pharmaceutical composition can be delivered using any drug delivery device.

[0188] The present disclosure includes the use of pharmaceutically acceptable salts of Spink9 polypeptides in the compositions and methods of the present invention. The disclosure also includes the use of buffer salts in compositions comprising Spink9 polypeptides in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, citric acid, acetic acid, alkyl, dialkyl, trialkyl, or tetra-alkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. Salts suitable for use with therapeutic polypeptides are known in the art (Sikora et al., (2020) Pharmaceuticals, 13, 442). In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid , gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0189] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0190] Useful dosages of the SPINK9 polypeptides disclosed herein can be determined, at least initially, by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art. The amount of SPINK9 polypeptide can be varied to obtain an amount effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0191] The dose of SPINK9 polypeptide administered will depend on a variety of factors, including the activity of the particular polypeptide, the route of administration, the time of administration, the severity of the disease, the rate of excretion, the duration of treatment, other active agents administered in combination, the age, sex, weight, condition, general health, and previous medical history of the subject being treated, and similar factors well known in the medical arts.

[0192] A physician of ordinary skill in the art can determine and prescribe the amount of pharmaceutical composition required. For example, a physician can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A larger total dose can be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0193] In certain embodiments, pharmaceutical compositions for administration, particularly subcutaneous and intravenous administration, may comprise from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 15 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 8 mg, from about 0.1 mg to about 6 mg, from about 0.1 mg to about 4 mg, from about 0.1 mg to about 2 mg, from about 0.1 mg to about 1 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 40 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 15 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 8 mg, from about 0.5 mg to about 6 mg, from about 0.5 mg to about 4 ... The doses include doses of Spink9 polypeptide of 0.5 mg to about 2 mg, about 1 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 15 mg, about 1 mg to about 10 mg, about 1 mg to about 8 mg, about 1 mg to about 6 mg, about 1 mg to about 4 mg, about 1 mg to about 2 mg, about 5 mg to about 50 mg, about 5 mg to about 40 mg, about 5 mg to about 30 mg, about 5 mg to about 25 mg, about 5 mg to about 20 mg, about 5 mg to about 15 mg, about 5 mg to about 10 mg, about 5 mg to about 8 mg, about 10 mg to about 50 mg, about 10 mg to about 40 mg, about 10 mg to about 30 mg, about 10 mg to about 25 mg, about 10 mg to about 20 mg, or about 10 mg to about 15 mg.

[0194] The dose of Spink9 polypeptide administered, particularly for subcutaneous and intravenous administration, may also be expressed in μg / kg. Exemplary doses of Spink9 polypeptide in μg / kg are about 1 μg / kg to about 500 μg / kg, about 1 μg / kg to about 250 μg / kg, about 1 μg / kg to about 100 μg / kg, about 1 μg / kg to about 50 μg / kg, about 1 μg / kg to about 25 μg / kg, about 1 μg / kg to about 15 μg / kg, and about 1 μg / kg to about 10 μg / kg. / kg, about 1μg / kg to about 5μg / kg, about 5μg / kg to about 500μg / kg, about 5μg / kg to about 250μg / kg, about 5μg / kg to about 100μg / kg , about 5μg / kg to about 50μg / kg, about 5μg / kg to about 25μg / kg, about 5μg / kg to about 15μg / kg, about 5μg / kg to about 10μg / kg, about 15μ g / kg~about 500μg / kg, about 15μg / kg~about 250μg / kg, about 15μg / kg~about 100μg / kg, about 15μg / kg~about 50μg / kg, about 1 5μg / kg to about 25μg / kg, about 50μg / kg to about 500μg / kg, about 50μg / kg to about 250μg / kg, about 50μg / kg to about 100μg / kg, The ranges are about 150 μg / kg to about 500 μg / kg, about 150 μg / kg to about 250 μg / kg, about 200 μg / kg to about 500 μg / kg, about 250 μg / kg to about 350 μg / kg, about 300 μg / kg to about 500 μg / kg, about 300 μg / kg to about 400 μg / kg, and about 400 μg / kg to about 500 μg / kg. Furthermore, the dose in μg / kg can be calculated by dividing the fixed doses described herein by 60-70 kg for adult subjects and 15-50 kg for pediatric subjects.

[0195] In certain embodiments, the Spink9 polypeptide, alone or as part of a pharmaceutical composition, is administered according to a course of treatment, which may extend over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 16 months, 24 months, 36 months, 48 months, 60 months or more. In certain embodiments, the course of treatment spans the remaining lifespan of the subject.

[0196] A subject may receive a single course of treatment or multiple (i.e., 1, 2, 3, 4, 5, or more) courses of treatment. When a subject receives multiple courses of treatment, such multiple courses of treatment may be consecutive (e.g., a second course of treatment begins immediately after the completion of a first course of treatment), or such multiple courses of treatment may be discontinuous and separated by non-treatment intervals (e.g., a third course of treatment begins one month after the completion of a second course of treatment). A combination of consecutive and non-continuous courses of treatment may also be used (e.g., a third course of treatment begins one month after the completion of a second course of treatment, and a second course of treatment begins immediately after the end of a first course of treatment). In certain embodiments, the non-treatment interval is 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0197] Dosage regimens can be used with courses of treatment. When multiple courses of treatment are administered to a subject, the dosage regimen can be the same for each course of treatment, or the dosage regimen can be different for one or more courses of treatment. In one embodiment, the dosing regimen comprises administering the Spink9 polypeptide during a course of treatment about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about every 7 days, about every 8 days, about every 9 days, about every 10 days, about every 11 days, about every 12 days, about every 13 days, about every 14 days, about every 15 days, about every 16 days, about every 17 days, about every 18 days, about every 19 days, about every 20 days, about every 21 days, about every 22 days, about every 23 days, about every 24 days, about every 25 days, about every 26 days, about every 27 days, about every 28 days, about every 29 days, about every 30 days, about every 45 days, about every 60 days, about every 75 days, about every 90 days, about every 105 days, or about every 120 days.

[0198] Preferably, the subject undergoes multiple courses of treatment. In certain embodiments, Spink9 polypeptide is administered by parenteral administration. In certain embodiments, Spink9 polypeptide is administered by subcutaneous administration. In certain embodiments, Spink9 polypeptide is administered by intravenous administration.

[0199] When a subject receives a course of treatment by subcutaneous administration, the Spink9 polypeptide can be injected into the subject at the same site (e.g., the upper arm) for each dose of the dosing regimen, or at different sites (e.g., the upper arm and the abdomen) for one or more doses of the dosing regimen. In certain embodiments, subcutaneous administration is achieved through the use of an implantable device.

[0200] In certain embodiments, the SPINK9 polypeptides disclosed herein can be used alone or administered in combination with another type of therapeutic agent, e.g., other agents useful for treating or preventing diseases or conditions characterized by abnormal kallikrein activity. The SPINK9 polypeptides disclosed herein can also be administered in combination with other therapeutic agents, e.g., other agents useful for treating or preventing diseases or conditions characterized by abnormal KLK5 activity. In certain embodiments, the SPINK9 polypeptides disclosed herein can also be administered in combination with one or more inhibitors of KLK family members, particularly KLK family members downstream of KLK5 (such as, but not limited to, KLK7 and KLK14). In certain embodiments, the SPINK9 polypeptides disclosed herein can also be administered in combination with one or more other therapeutic agents useful for treating or preventing Netherton syndrome. For example, without being bound by theory or methodology, "co-administration" can refer to any form of administration of a SPINK9 polypeptide disclosed herein with another therapeutic agent, such that the SPINK9 polypeptide or other therapeutic agent is administered while the previously administered agent (e.g., the SPINK9 polypeptide or other therapeutic agent) is still effective in the body (e.g., the SPINK9 polypeptide and the other therapeutic agent are effective in the patient simultaneously, which may include a synergistic effect). The SPINK9 polypeptide and the other different therapeutic compound can be administered either simultaneously or sequentially, in either the same formulation or in separate formulations. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic agents. [Example]

[0201] The invention has now been generally described and will be more readily understood by reference to the following examples, which are included solely for purposes of illustration of certain aspects and embodiments of the invention and are not intended to limit the invention.

[0202] Example 1: Vector construction and expression of SPINK9 polypeptide SPINK9 polypeptides were recombinantly produced as follows. First, a cDNA sequence encoding the desired SPINK9 domain (e.g., amino acids 20-86 of SEQ ID NO: 1) fused to a C-terminal IgG Fc fragment was designed. Codon usage was optimized for mammalian expression systems using the GeneArt codon optimizer, and the proposed Kozak sequence was used. cDNA was synthesized, and the nucleotide sequence was confirmed. The cDNA was cloned into the pcDNA3.4 expression vector using the EcoR1 and HindIII sites. The pcDNA3.4 vector uses the full-length human cytomegalovirus (CMV) immediate-early promoter for high-level gene expression, along with a woodchuck posttranscriptional regulatory element (WPRE) downstream of the cloning site, to enhance transcriptional expression. To ensure secretion into the cell culture medium, a mouse IgG heavy chain signal peptide (MGWSCIILFLVATATGVHS) or wild-type SPINK9 signal peptide (e.g., amino acids 1-19 of SEQ ID NO: 1) was added at the N-terminus of the SPINK9 polypeptide. The signal peptide was cleaved during translocation. To express SPINK9 polypeptide, Expi293 cells (A14527, ThermoFisher) or CHO-S cells (R80007, ThermoFisher) were used. For expression, Expi293 cells were transiently transfected with the expression vector, and the cell culture was harvested. SPINK9 polypeptide was purified from cell culture medium using a HiTrap Protein G HP 5 ml column (17-0405-01, GE Healthcare). The filtered cell culture medium was loaded onto a Protein G column according to the Protein G protocol, using PBS (binding buffer) to equilibrate the column and 0.7% acetic acid (elution buffer) to elute the SPINK9 polypeptide. Elution was monitored by measuring absorbance at 280 nm. After purification, SPINK9 polypeptide was stored in PBS; therefore, immediately after elution, the buffer was exchanged by either dialysis or a desalting column with the appropriate molecular weight cutoff. SPINK9 polypeptide was concentrated to a concentration of 1 mg / ml using an Amicon ultracentrifugal filter (Merck Millipore) with a molecular weight cutoff of 50 kDa.Protein concentrations were measured on a microvolume spectrophotometer using the A280 setting. Expression of the desired SPINK9 polypeptide was further confirmed by SDS page and Western blot analysis under reducing and non-reducing conditions.

[0203] Example 2: Evaluation of the inhibitory activity of SPINK9 polypeptides against KLK5. The effect of SPINK9 polypeptide on KLK5 enzyme activity was evaluated. Assays were performed by incubating 20 nM KLK5 in 100 mM NaH2PO4, pH 7.5 and pH 5.5, 1 mM CHAPS with SPINK9 polypeptide (half-log dilution series from 0.3 nM to 300 nM) or assay buffer as a control for 10 minutes at 22°C. The reaction was then initiated by the addition of 100 μM of the substrate BOC-VPR-AMC (R&D Systems, Minneapolis, Minnesota) in a total reaction volume of 100 μL. After a 15-minute incubation, the plate was excited at 340 nm and fluorescence emission was measured at 460 nM using a Synergy Neo microplate reader (BioTek®, Winooski, Vermont). The 50% maximal inhibitory concentration (IC) was determined. 50 ) was calculated by nonlinear regression analysis using a four-parameter, sigmoidal, dose-response equation using Prism GraphPad (GraphPad Software, San Diego, California). The results are shown in Table 9. Wild-type Spink9 (SEQ ID NO: 2) binding to KLK5 had IC5 of 80 nM and 685 nM at pH 7.5 and 5.5, respectively. 50 showed. [Table 9]

[0204] Example 3: Evaluation of the inhibitory activity of SPINK9 polypeptides against KLK7 and KLK14. The effect of SPINK9 polypeptide on KLK7 and KLK14 enzyme activity was evaluated. Both rhKLK7 and rhKLK14 were obtained as zymogens and each required activation with thermolysin. rhKLK7 or rhKLK14 was diluted to 200 mg / mL in activation buffer (50 mM Tris, 10 mM CaCl, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 7.5) and mixed with an equal volume of 20 mg / mL thermolysin in the same buffer. Activation was carried out at 37°C for 1 hour (rhKLK14) or 2 hours (rhKLK7), and the reaction was stopped by the addition of 50 mM EDTA.

[0205] The KLK14 assay was performed as described in Example 2 above, except that the concentration of active KLK14 was 0.2 mg / ml. Enzyme activity was measured by fluorescence and expressed as IC 50 was calculated as described in Example 2 above.

[0206] The KLK7 assay was performed by incubating 2 mg / mL of active KLK7 in 50 mM Tris, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 8.0, with half-log dilutions (half-log dilution series from 0.3 nM to 300 nM) of SPINK9 polypeptide or assay buffer as a control for 15 min at 22 °C. The reaction was initiated by the addition of 20 mM of the substrate MCA-Arg-Pro Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2 (R&D Systems) in a total reaction volume of 100 mL. After the 15-minute incubation, the plate was excited at 320 nm and fluorescence emission was measured at 405 nM using a Synergy Neo microplate reader (BioTek®, Winooski, Vermont). IC 50 was calculated as described in Example 2 above.

[0207] The results for both KLK7 and KLK14 are shown in Table 10. [Table 10]

[0208] Example 4: Evaluation of human serum stability. An exemplary SPINK9 polypeptide, SPINK9-112.2K62G.Fc (SEQ ID NO: 76), was diluted (1:9) in freshly collected human serum and distributed into 200 μL aliquots. Five aliquots of the sample were then kept in an incubator set at 37°C and 5% CO2. Samples were collected and flash-frozen on days 0, 1, 4, 7, and 14, and then kept at -80°C until analysis. The binding activity of the tested samples to recombinant human KLK5 C-6His (Novoprotein, catalog number C415) was assessed by ELISA as described below.

[0209] The binding of SPINK9-112.2K62G.Fc after serum incubation was determined by ELISA. Plates were pre-coated with 100 μL / well of THE™ His-tag mAb (2 μg / mL) (Genscript, Catalog No.: A00186) overnight in a refrigerator set at 4°C. After blocking with 200 μL / well of 0.5% casein for 1 hour, the plates were washed three times with 1×PBST. Then, 0.25 μg / mL of recombinant human KLK5 C-6His was added to the plate at a volume of 100 μL / well and incubated at ambient temperature for 1 hour. After washing the plate three times with 1x PBST, SPINK9-112.2K62G.Fc at various concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.0013, 0.00026, 0.000051, and 0.000010 nM) containing dilution buffer (0.25% casein) was added to the plate in a volume of 100 μL / well and incubated at ambient temperature for 2 hours. After washing the plate three times with 1x PBST, 100 μL / well of HRP-labeled goat anti-human IgG antibody (1:5000) (Bethyl, Cat. No.: A80-304P-16) containing dilution buffer (0.25% casein) was added to each well and incubated at ambient temperature for 1 hour. After washing six times with 1x PBST, color development was performed by dispensing 100 μL / well of TMB substrate (Life Technologies, Cat. No.: 00203) for 4 minutes, and the reaction was stopped by adding 100 μL / well of 2M HCl. Absorbance was read at 450 and 540 nm using a microplate reader (SpectraMax M5e). EC 50 Values were calculated by four-parameter nonlinear regression analysis using GraphPad Prism software.

[0210] Human serum-incubated SPINK9-112.2K62G.Fc was tested for its ability to bind to recombinant human KLK5 C-6His at different time points. The results of the serum stability study (Table 11) show that SPINK9-112.2K62G.Fc incubated with serum for up to two weeks maintained its binding activity to KLK5. [Table 11]

[0211] Table 12 shows human serum stability and affinity studies for other Spink9 polypeptides performed according to the procedures described above. [Table 12]

[0212] Example 5: Binding to human KLK5 at pH 7.4 by surface plasmon resonance (SPR). SPR analysis was performed using a Biacore T200 (GE Healthcare) equipped with a Series S CM5 sensor chip (GE Healthcare). Anti-human IgG Fc monoclonal antibody was diluted to 25 μg / ml in immobilization buffer (10 mM sodium acetate, pH 5.0).

[0213] A CM5 sensor chip was activated with an activator mixture (prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection) at a flow rate of 10 μL / min for 420 seconds. Then, 25 μg / mL of anti-human IgG Fc monoclonal antibody in immobilization buffer was injected into the Fc2 sample channel at a flow rate of 10 μL / min for 420 seconds, resulting in an immobilization level of approximately 7000 RU. The sensor chip was deactivated with 1 M ethanolamine hydrochloride-NaOH (GE Healthcare) at a flow rate of 10 μL / min for 420 seconds. The reference surface channel, Fc1, was prepared in the same manner.

[0214] Spink9 polypeptide was diluted to 0.1–0.7 μg / mL in running buffer (1× HBS-EP, containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20, pH 7.4) and then injected into channel Fc2 at a flow rate of 10 μL / min to reach capture levels of approximately 35.6–64.5 RU.

[0215] KLK5 protein (Novoprotein, catalog no. C415) was diluted to five or six concentrations in running buffer (1x HEPES, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) as described below. KLK5 protein was injected into the Fc1 and Fc2 channels at a flow rate of 30 μL / min for a 120-second association phase, followed by a 300-second dissociation (all association and dissociation phases were performed in running buffer). Increasing concentrations of KLK5 analyte were analyzed. After each cycle of interaction analysis, the sensor chip surface was regenerated with 3 M MgCl as injection buffer at a flow rate of 10 μL / min for 30 seconds to remove the ligand and any unbound KLK5. Spink9 WT-100, 50, 25, 12, 5, and 6.25 nM KLK5 Spink9-30.Fc (SEQ ID NO: 55) - 65, 32.5, 16.25, 8.125, 4.063, and 2.032 nM KLK5 Spink9-112.K62G.Fc (SEQ ID NO: 56) - 50, 25, 12, 5, 6.25, and 3.125 nM KLK5 Spink9-183.Fc (SEQ ID NO: 57) - 100, 50, 25, 12, 5, 6.25, and 3.125 nM KLK5 Spink9-186.Fc (SEQ ID NO: 58) - 100, 50, 25, 12, 5, 6.25, and 3.125 nM KLK5 Spink9-189.Fc (SEQ ID NO: 59) - 100, 50, 25, 12, 5, 6.25, and 3.125 nM KLK5 Spink9-236.Fc (SEQ ID NO: 60) - 100, 50, 25, 12, 5, 6.25, and 3.125 nM KLK5

[0216] The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. The experimental data were fitted by a steady-state affinity model. The molar concentration of human KLK5 was calculated using a molecular weight of 30.65 kDa. Table 13 shows the K values for the tested polypeptides. D Indicates the value. [Table 13]

[0217] Example 6: Binding to human FcRn at pH 6.0 and 7.4 by surface plasmon resonance (SPR). SPR analysis was performed using a Biacore 8K equipped with a series S CM5 sensor chip (Cytiva). The CM5 sensor chip was activated with an activator mixture (prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection) at a flow rate of 10 μL / min for 420 seconds. Then, 10 μg / mL of Spink9-30 (SEQ ID NO: 55), Spink9-112.K62G (SEQ ID NO: 56), Spink9-183 (SEQ ID NO: 57), Spink9-186 (SEQ ID NO: 58), Spink9-189 (SEQ ID NO: 59), Spink9-236 (SEQ ID NO: 60), Spink9-112.2K62G (SEQ ID NO: 76), and rituximab (Roche) in 10 mM NaAc (pH 5.5) were injected into Fc2 at a flow rate of 10 μL / min for 60 seconds. The reference channel, Fc1, was blocked. The sensor chip was deactivated with 1 M ethanolamine-HCl (Cytiva) for 420 seconds at a flow rate of 10 μL / min.

[0218] Eight concentrations of the analyte human FcRn (46.875, 93.75, 187.5, 375, 750, 1500, 3000, and 6000 nM) in 1x PBST (50 mM NaHPO / NaHPO, 150 mM NaCl, 0.05% Tween-20, pH 6.0 or 7.4) (AcrobioSystems, catalog number: FCM-H5286) and running buffer (1x PBST, pH 6.0) were injected into Fc1 and Fc2 at a flow rate of 30 μL / min for a 60-second association phase, followed by a 90-second dissociation phase. 1x PBS (pH 7.4) was injected into the flow cell as a regeneration buffer for 45 seconds at a flow rate of 10 μL / min after each dissociation phase.

[0219] The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. The experimental data were fitted by a steady-state affinity model. The molar concentration of human FcRn was calculated using a molecular weight of 45 kDa. Table 14 shows the K values for the tested polypeptides. D Indicates the value. [Table 14]

[0220] Example 7: Single-dose PK study of SPINK9 polypeptide in cynomolgus monkeys. Eight cynomolgus monkeys (four males and four females) weighing between 2.5 and 3.5 kg were used in this study. The animals were fed approximately 120 g of certified monkey chow daily (Beijing Vital Keao Feed Co., Ltd. Beijing, PR China). The monkeys received fresh fruit daily. Reverse osmosis purified water was provided to the animals ad libitum via an automated watering system.

[0221] Four male and four female cynomolgus monkeys were randomly assigned to four groups (one male and one female monkey per group) to evaluate the pharmacokinetics and pharmacodynamics of Spink9-112.K62G (SEQ ID NO: 56), Spink9-112.2K62G.Fc (SEQ ID NO: 76), Spink9-186 (SEQ ID NO: 58), and Spink9-183 (SEQ ID NO: 57). Spink9 polypeptides were administered at a single dose of 10 mg / kg via intravenous (IV) infusion over 45 minutes in PBS buffer. Animals in Group 1 were administered Spink9-112.K62G (SEQ ID NO: 56). Animals in Group 2 were administered Spink9-112.2K62G.Fc (SEQ ID NO: 76). Animals in Group 3 were administered Spink9-186 (SEQ ID NO: 58). Animals in group 4 were administered Spink9-183 (SEQ ID NO: 57). Animals were randomly assigned to groups based on body weight. Animals were weighed prior to dose administration and doses were determined based on body weight recorded on the dose record sheet. The start date of the first dose was recorded as day 0.

[0222] At least 200 μL of blood samples were collected from the cephalic or saphenous vein from animals in all groups at each sampling time point. For samples collected within the first hour of dosing, ±1 minute was acceptable. For the remaining time points, samples were collected within 5% of the scheduled time. All blood samples were collected in commercially available tubes containing a coagulant. The tubes containing the blood samples were then kept at room temperature for 30 minutes before centrifugation. The samples were centrifuged, and serum was collected for PK assay. Samples were collected at the following time points: pre-dose (day -1), and 5 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 12 hours, 24 hours (day 1), 48 hours (day 2), 96 hours (day 4), 144 hours (day 6), 192 hours (day 8), 240 hours (day 10), 288 hours (day 12), 384 hours (day 16), 480 hours (day 20), 576 hours (day 24), and 672 hours (day 28) post-dose.

[0223] Animals were observed (twice daily) for weight and feeding status. Cageside observations for general health and appearance were performed once daily. Animals were observed before and after dosing and at each sample collection time point. Weight and feeding status were recorded. Each animal was weighed twice during the acclimation period and once pre-test (day 0). After test article administration, each animal was weighed twice per week from days 0 to 28. Daily food consumption was also measured for all animals from days 0 to 28.

[0224] PK method Concentrations of test articles in serum were determined using two different assay bioanalytical ELISA methods, the Fc+Fc assay and the KLK5+Fc assay.

[0225] Fc+Fc: Briefly, 96-well ELISA plates were incubated overnight at 4°C with a 1.0 μg / mL solution of goat anti-human IgG (Southern Biotech, catalog no. 2049-01). After washing and blocking, serially diluted plasma samples were added, followed by biotin-labeled goat anti-human IgG (dilution factor: 8000) (Southern Biotech, catalog no. 2049-01) as the detection antibody. Streptavidin-HRP (dilution factor: 20000) (Thermo-Fischer, catalog no. 21127) and TMB (Life Technologies, catalog no. 002023) substrate were used for color development. The reaction was stopped after approximately 5–10 min by the addition of 2 M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax M5e). Using a standard curve, the OD values of the samples were used to obtain the plasma concentrations of the test articles. The detection limit of this ELISA method was 0.391 ng / mL.

[0226] KLK5+Fc: Briefly, a 96-well ELISA plate was incubated overnight at 4°C with a 1.0 μg / mL His-tag antibody (Sino Biological, catalog number: A00186-100). The plate was washed, and 0.25 μg / mL recombinant human KLK5 C-6His (Novoprotein, catalog number: C415) was added to each well. After washing and blocking, serially diluted plasma samples were added, followed by biotin-labeled goat anti-human IgG (dilution factor: 8000) (Southern Biotech, catalog number: 2049-08). Streptavidin-HRP (dilution factor: 20000) (Thermo-Fischer, catalog number: 21127) and TMB (Life Technologies, catalog number: 002023) substrate were used for color development. The reaction was stopped after approximately 5–10 min by the addition of 2 M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax M5e). Using the standard curve, the OD values of the samples were used to obtain the plasma concentrations of the test articles. The detection limit of this ELISA method was 0.195 ng / mL.

[0227] The serum concentrations of the four test articles in monkeys were subjected to non-compartmental pharmacokinetic analysis by using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal rule was applied to obtain PK parameters.

[0228] Pharmacokinetic parameters for the test articles are provided in Tables 15 and 16 for the Fc+Fc and KLK5+Fc methods, respectively, and are shown in Figures 2 and 3, respectively. [Table 15] [Table 16]

[0229] All animals behaved normally. No formulation-related effects were observed on body weight and weight gain (Figure 1). No formulation-related effects were observed on food consumption during the study.

[0230] Pharmacokinetic results showed a mean half-life of 128 h in the Fc+Fc assay and a mean AUC of 8158 h·μg / mL. 0-last , and a mean Cl of 1.14 mL / h / kg _obs , as well as a mean half-life of 131 h and a mean AUC of 8114 h·μg / mL in the KLK5+Fc assay. 0-last , and a mean Cl of 1.03 mL / h / kg _obs The results show that the Spink9-112.2K62G.Fc polypeptide showed the most favorable results.

[0231] equivalent While specific embodiments of the subject invention have been discussed, the foregoing specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with the specification, along with their full scope of equivalents, and such variations.

[0232] Incorporation by Reference All patent applications, patents, and publications cited herein are incorporated herein by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and except to the extent that the incorporated material contradicts the express disclosure of this specification, in which case the language of this disclosure will control.

Claims

1. A polypeptide comprising at least one mutant SPINK9 domain, wherein the at least one mutant SPINK9 domain comprises, in order from the N-terminus to the C-terminus, a first amino acid sequence sharing at least 70% sequence identity with amino acids 1 to 20 shown in SEQ ID NO: 2, a second amino acid sequence comprising at least four amino acid substitutions with amino acids 21 to 34 shown in SEQ ID NO: 2, and a third amino acid sequence sharing at least 70% sequence identity with amino acids 35 to 67 shown in SEQ ID NO:

2.

2. The second amino acid sequence contains at least one amino acid substitution at positions 22, 24, 25, 26, 28, 29, 30, or any combination thereof of SEQ ID NO: 2, or The first amino acid sequence optionally includes the amino acids at positions 1, 2, and 3 of SEQ ID NO: 2, and the second amino acid sequence includes at least one amino acid substitution at positions 22, 24, 25, 26, 28, 29, 30, or any combination thereof of SEQ ID NO: 2, or The polypeptide according to claim 1, wherein the first amino acid sequence includes deletions of the amino acids at positions 1, 2, and 3 of SEQ ID NO: 2, and the second amino acid sequence includes at least one amino acid substitution at positions 22, 24, 25, 26, 28, 29, 30, or any combination thereof of SEQ ID NO:

2.

3. The polypeptide according to claim 2, wherein the second amino acid sequence further comprises one or more amino acid changes at positions 21, 23, 27, 31, 32, 33, and 34 of SEQ ID NO:

2.

4. The polypeptide according to claim 2, wherein the second amino acid sequence further comprises one or more amino acid changes at positions 21, 31, 32, 33, and 34 of SEQ ID NO:

2.

5. The second amino acid sequence is, i) The amino acid at position 22 of SEQ ID NO: 2 includes substitution with the amino acid Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His, or the amino acid at position 22 of SEQ ID NO: 2 is Gly. ii) The amino acid at position 24 of SEQ ID NO: 2 includes substitution with the amino acid Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr, or the amino acid at position 24 of SEQ ID NO: 2 is Glun. iii) The amino acid at position 25 of SEQ ID NO: 2 includes substitution with the amino acid Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met, or the amino acid at position 25 of SEQ ID NO: 2 is Arg. iv) The amino acid at position 26 of SEQ ID NO: 2 contains a substitution with the amino acid Tyr, Trp, Met, His, Lys, or Asn, or the amino acid at position 26 of SEQ ID NO: 2 is Phe. v) The amino acid at position 28 of SEQ ID NO: 2 contains a substitution with a Thr, Ser, or Gly amino acid, or the amino acid at position 28 of SEQ ID NO: 2 is His. vi) The amino acid at position 29 of SEQ ID NO: 2 includes a substitution with an Arg or Lys amino acid, or the amino acid at position 29 of SEQ ID NO: 2 is His, or vii) The polypeptide according to claim 1, wherein the amino acid at position 30 of SEQ ID NO: 2 is substituted with a Glu or Asp amino acid, or the amino acid at position 30 of SEQ ID NO: 2 is Met.

6. The polypeptide according to claim 1, wherein the third amino acid sequence includes a substitution of the amino acid at position 43 of SEQ ID NO: 2 with a glycerin amino acid.

7. The polypeptide according to claim 1, wherein the at least one mutant SPINK9 domain comprises an amino acid sequence selected from SEQ ID NOs: 4-23 or 115-134.

8. The polypeptide according to claim 1, wherein the polypeptide comprises independently selected first and second mutant SPINK9 domains, each of the first and second mutant SPINK9 domains comprising one independently selected amino acid sequence from SEQ ID NOs: 4-23 or 115-134.

9. The polypeptide according to claim 1, comprising a first polypeptide chain and a second polypeptide chain linked via an Fc moiety, wherein the first polypeptide chain comprises first and second mutant SPINK9 domains, and the second polypeptide chain comprises third and fourth mutant SPINK9 domains, and each of the first, second, third, and fourth mutant SPINK9 domains comprises an independently selected amino acid sequence of SEQ ID NOs: 4-23 or 115-134.

10. A polypeptide comprising at least one mutant SPINK9 domain, wherein the at least one mutant SPINK9 domain is of formula I: ______________________________________________________________________ ____________________________________________________________________________ １ ︌n️ ２ ︸ ３ ︸ ４ y) ︸ ５ ︸ ６ ︸ ７ ________________________________________________________ () ８ ______________________________________________________________ (r) sp !ly ___________________________________ It includes the amino acid sequence represented by, however, the amino acid sequence represented by formula I is not sequence number 2, During the ceremony, X1 is selected from i) Gly, Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; ii) Gly, Glu, or Met; iii) Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; or iv) Glu or Met. X2 is selected from i) Gln, Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; ii) Gln, Glu, or Thr; iii) Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; or iv) Glu or Thr, X3 is selected from i) Arg, Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; ii) Arg, Leu, Ile, His, Glu, or Trp; iii) Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; or iv) Leu, Ile, His, Glu, or Trp. X4 is selected from i) Phe, Tyr, Trp, Met, His, Lys, or Asn; ii) Phe, Tyr, or Trp; iii) Tyr, Trp, Met, His, Lys, or Asn; or iv) Trp or Tyr. X 5 is selected from i) His, Thr, Ser, or Gly; ii) His or Thr; iii) Thr, Ser, or Gly; or iv) Thr, X 6 is selected from i) His, Arg, or Lys; ii) His or Arg; iii) Arg or Lys; or iv) Arg. X7 is selected from i) Met, Glu or Asp; ii) Met or Glu; iii) Glu or Asp; or iv) Glu, and X 8 is a polypeptide selected from i) Lys or Gly; ii) Lys; or iii) Gly.

11. X ５ -X ６ -X ７ is the polypeptide according to claim 10, selected from the group consisting of Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, and Gly-Arg-Asp.

12. The polypeptide according to claim 10, wherein the polypeptide comprises 2 to 20 independently selected mutant SPINK9 domains, each mutant SPINK9 domain comprising one independently selected amino acid sequence of formula I or sequence numbers 4 to 7 or 9 to 23.

13. The polypeptide according to claim 10, wherein the polypeptide comprises independently selected first and second mutant SPINK9 domains, each of the first and second mutant SPINK9 domains comprising one independently selected amino acid sequence of formula I or sequence numbers 4-7 or 9-23.

14. The polypeptide according to claim 10, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain linked via an Fc moiety, the first polypeptide chain comprises first and second mutant SPINK9 domains, the second polypeptide chain comprises third and fourth mutant SPINK9 domains, and each of the first, second, third, and fourth mutant SPINK9 domains comprises one independently selected amino acid sequence of formula I or sequence numbers 4-7 or 9-23.

15. The polypeptide according to claim 10, comprising a first polypeptide chain and a second polypeptide chain linked via an Fc moiety, wherein the first polypeptide chain comprises a first mutant SPINK9 domain, and the second polypeptide chain comprises a second mutant SPINK9 domain, and each of the first and second mutant SPINK9 domains comprises one independently selected amino acid sequence of formula I or sequence numbers 4-7 or 9-23.

16. A polypeptide comprising at least one mutant SPINK9 domain, wherein the at least one mutant SPINK9 domain is of formula IV: ︸ ９ ︸ １０ ︸ １１ ___________________________________________________________________ ____________________________________________________________________________ １ ︌n️ ２ ︸ ３ ︸ ４ y) ︸ ５ ︸ ６ ︸ ７ ________________________________________________________ () ８ ______________________________________________________________ (r) sp !ly _________________________________________ It includes an amino acid sequence represented by, however, the amino acid sequence represented by formula IV is not SEQ ID NO: 2, X1 is selected from i) Gly, Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; ii) Gly, Glu, or Met; iii) Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys, or His; or iv) Glu or Met. X2 is selected from i) Gln, Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; ii) Gln, Glu, or Thr; iii) Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser, or Tyr; or iv) Glu or Thr, X3 is selected from i) Arg, Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; ii) Arg, Leu, Ile, His, Glu, or Trp; iii) Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp, or Met; or iv) Leu, Ile, His, Glu, or Trp. X4 is selected from i) Phe, Tyr, Trp, Met, His, Lys, or Asn; ii) Phe, Tyr, or Trp; iii) Tyr, Trp, Met, His, Lys, or Asn; or iv) Trp or Tyr. X 5 is selected from i) His, Thr, Ser, or Gly; ii) His or Thr; iii) Thr, Ser, or Gly; or iv) Thr. X 6 is selected from i) His, Arg, or Lys; ii) His or Arg; iii) Arg or Lys; or iv) Arg. X 7 is selected from i) Met, Glu, or Asp; ii) Met or Glu; iii) Glu or Asp; or iv) Glu, X 8 is selected from i) Lys or Gly; ii) Lys; or iii) Gly, X 9 is selected from i) absent or ii) Ile, X 10 is selected from i) absent, or ii) Glu, and X 11 is a polypeptide selected from i) absent or ii) Cys.

17. X ５ -X ６ -X ７ The polypeptide according to claim 16, wherein is selected from the group consisting of Thr-Arg-Glu, Thr-Arg-Asp, Thr-Lys-Glu, Thr-Lys-Asp, Ser-Arg-Glu, and Gly-Arg-Asp.

18. The polypeptide according to claim 16, wherein the polypeptide comprises 2 to 20 independently selected mutant SPINK9 domains, each mutant SPINK9 domain comprising one independently selected amino acid sequence of formula IV or SEQ ID NOs: 115-118 or 120-134.

19. The polypeptide according to claim 16, wherein the polypeptide comprises independently selected first and second mutant SPINK9 domains, each of the first and second mutant SPINK9 domains comprising one independently selected amino acid sequence of formula IV or sequence numbers 115-118 or 120-134.

20. The polypeptide according to claim 16, wherein adjacent mutant SPINK9 domains are linked by direct fusion, a linker sequence, or any combination thereof.

21. The polypeptide according to claim 20, wherein at least one mutant SPINK9 domain is bound to an adjacent mutant SPINK9 domain by a linker sequence.

22. The polypeptide according to claim 20, wherein at least one mutant SPINK9 domain is bound to an adjacent mutant SPINK9 domain by direct fusion.

23. The polypeptide according to claim 20, wherein the linker sequence includes cleavage sites.

24. The polypeptide according to claim 23, wherein the cleavage site is recognized by a subtilisin-like proprotein convertase.

25. The polypeptide according to claim 16, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain linked via an Fc moiety, the first polypeptide chain comprises a first mutant SPINK9 domain, the second polypeptide chain comprises a second mutant SPINK9 domain, and each of the first and second mutant SPINK9 domains comprises one independently selected amino acid sequence of formula IV or sequence numbers 115-118 and 120-134.

26. The polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 114, or The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 55 to 114, in which amino acids 1 to 3 are deleted.

27. A nucleic acid encoding a polypeptide according to any one of claims 1 to 26.

28. A vector comprising the nucleic acid described in claim 27.

29. i) a nucleic acid encoding a polypeptide according to any one of claims 1 to 26, or ii) a host cell comprising a vector comprising a nucleic acid encoding a polypeptide according to any one of claims 1 to 26.

30. A method for producing a polypeptide comprising at least one mutant SPINK9 domain, comprising expressing the polypeptide in a host cell according to claim 29, and isolating the polypeptide.

31. A composition comprising a polypeptide according to any one of claims 1 to 26, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, or a host cell containing the nucleic acid or vector.

32. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

33. A composition for treating or preventing a disease or condition characterized by abnormal kallikrein activity, comprising a polypeptide according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof.

34. The composition according to claim 33, wherein the disease or condition is characterized by abnormal kallikrein-related peptidase 5 (KLK5) activity.

35. (i) The disease or condition is a skin disease, (ii) The skin disease is eczema, atopic eczema, atopic dermatitis, autosomal recessive ichthyosis, ichthyoid erythroderma, psoriasis, UV-induced skin injury, rosacea, or a skin infection, (iii) The disease or condition is selected from the group consisting of: hypersensitivity of the immune system (atopic dermatitis), hyper IgE syndrome, allergies (including allergies to food and airborne substances), asthma, allergic asthma, chronic inflammation, rhinitis, conjunctivitis, angioedema, eosinophilia, eosinophilic esophagitis, growth retardation, growth disorders, trichorrhexis invaginata (TI), respiratory tract infections, systemic infections, and gastrointestinal disorders, or (iv) The disease or condition is cancer, (v) The composition according to claim 34, wherein the cancer is ovarian cancer, uterine cancer, colorectal cancer, urothelial carcinoma of the bladder, oral squamous cell carcinoma, breast cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, head and neck cancer, glioma multiforme, or neuroblastoma.

36. The composition according to claim 34, wherein the disease or condition is Netherton syndrome.

37. The polypeptide according to any one of claims 1 to 26, wherein the polypeptide binds to and inhibits an active kallikrein-related peptidase.

38. The polypeptide according to any one of claims 1 to 26, wherein the polypeptide inhibits the activity of kallikrein-related peptidase 5 (KLK5).

39. The polypeptide according to claim 38, wherein the inhibition is selective for or specific to KLK5.

40. A polypeptide according to any one of claims 1 to 26, further comprising a signal sequence.

41. The polypeptide according to claim 40, wherein the signal sequence is bonded to the amino(N)-terminus of the polypeptide by direct fusion or by a linker sequence.

42. The polypeptide according to claim 40, wherein the signal sequence is bonded to the carboxy(C)-terminus of the polypeptide by direct fusion or by a linker sequence.

43. The polypeptide according to any one of claims 1 to 26, further comprising an immunoglobulin Fc region or a functional fragment thereof.

44. The polypeptide according to claim 43, wherein the immunoglobulin Fc region or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 54.

45. The immunoglobulin Fc region or its functional fragment is given by formula II: Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro X １ Cys Pro Ala Pro X ２ X ３ X ４ Gly X ５ Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu X ６ Ile X ７ Arg X ８ Pro Glu Val Thr Cys Val Val X ９ Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr X １０ Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val X １１ His X １２ Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys X １３ Leu Pro X １４ X １５ Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Gln Vau Tyr Thr Leu Alu Aor A. Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu T`r 5es Leu Thr LeuAsp Ser Cys Ser Val X １６ His Glu Ala Leu ^ １７ X １８ His Tyr Thr Gln Lys Ser Leu�ro Gly Ly It contains the amino acid sequence, In the formula, X １ is Pro or Ser, X ２ , is Glu or Pro, X ３ and X ４ Each is independently Lue or Ala, and X ５ is Gly or Ala, X ６ is Met or Tyr, X ７ is either Ser or Thr, and X ８ is either Thr or Glu, and X ９ is Val or Glu, and X １０ is Asn or Ala, X １１ is Leu or Asp, X １２ is either GLN or His, and X １３ is Ala or Gly, X １４ is Ala or Ser, X １５ is Pro or Ser, X １６ is Met or Leu, X １７ is His or Lys, X １８ The polypeptide according to claim 43, wherein is Asn, Ser, or Phe.

46. The polypeptide according to claim 43, wherein the immunoglobulin Fc region or a functional fragment thereof is bound to the mutant SPINK9 domain by direct fusion or by a linker sequence.

47. The polypeptide according to claim 1, comprising any one amino acid sequence of sequence numbers 55 to 114.

48. The polypeptide according to claim 1, comprising any one amino acid sequence of sequence numbers 55 to 60.

49. The polypeptide according to claim 1, comprising any one amino acid sequence of sequence numbers 75 to 80.

50. The polypeptide according to claim 1, comprising any one amino acid sequence of SEQ ID NOs. 95 to 100.

51. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO:

139.

52. A composition comprising the nucleic acid described in Claim 27.

53. A composition comprising the vector described in Claim 28.

54. A composition comprising the host cell described in Claim 29.

Images