Fusion protein inhibitors of KLK5

Abstract

The present invention discloses polypeptides useful, inter alia, for inhibiting or suppressing the activity of trypsin-associated proteases, and thus treating diseases and conditions involving a disorder of trypsin-associated protease activity. Pharmaceutical compositions and methods of treatment using the polypeptides and pharmaceutical compositions are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 390,780 filed on July 20, 2022. Background Art

[0003] KLKs are a family of 15 trypsin-like and chymotrypsin-like serine proteases. KLKs are secreted as zymogens and need to be activated by removing the terminal peptide portion via specific amino-terminal proteolysis. Some KLKs rely on activation by other KLKs or other proteases, while some KLKs, such as KLK5, can self-activate. Therefore, KLKs act via a proteolytic cascade in vivo. KLKs are widely expressed in a variety of tissues, including kidneys, brain, respiratory system, gastrointestinal tract, epidermis, and reproductive tract. KLKs generally regulate a variety of basic physiological functions in a cell-specific manner, including regulating immunity, inflammation, and cancer.

[0004] KLK plays an important role in the epidermis, among other roles. KLK5 and KLK7 degrade intercellular bridge proteins, a group of proteins responsible for the structural integrity of the epidermis. For example, old cells fall off the surface of the skin and maintenance of a healthy epidermis requires intercellular bridge degradation, a process known as desquamation (Simon et al. 2001; Caubet et al. 2004). Thus, KLK plays a role in maintaining the integrity of the skin barrier and preventing desquamation and inflammation. KLK5 is an important regulator of proteolytic activity in the epidermis, because KLK5 can not only activate its own zymogen, but also activate many other KLK family zymogens, and has been shown to activate proKLK7 (Caubet et al. 2004; Brattsand et al. 2005; Yoon et al. 2007). KLK5 also shows activation of metalloproteinases transmembrane peptidase (meprin)-α and transmembrane peptidase-β, which are associated with basal keratinocyte proliferation and differentiation processes (Ohler et al. 2010). In addition, KLK5 and KLK14 can activate protease activated receptor-2 (PAR2), a G protein-coupled receptor present on the membrane of many cell types (including keratinocytes), and play a regulatory role in the skin during inflammation (including the production of proinflammatory cytokines that lead to a proinflammatory environment, activation of Langerhans cells, and induction of pro-allergic Th2 cells), epidermal barrier function, and itch (Oikonomopoulou et al. 2006; Stefansson et al. 2008). KLK, including KLK5, is also involved in the processing of cathelicidin antimicrobial peptides that kill microorganisms, and regulates host immunity and cell growth responses (Lai and Gallo, 2009; Yamasaki et al. 2006; Eissa et al. 2011).

[0005] 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 patients with rosacea, who also display abnormally high levels of kesarin in their 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 expression, including atopic dermatitis and hay fever (Netherton, 1958; Burns et al. 2004).

[0006] However, treatment of diseases and conditions associated with increased activity of KLKs is largely non-specific and targets symptoms rather than the underlying cause of the disease, and causes undesirable side effects such as atrophy after long-term use of glucocorticoids. Therefore, there is a need to develop other KLK inhibitors, particularly KLK5 inhibitors, that have therapeutic potential in the treatment of many diseases and conditions. Summary of the invention

[0007] In one aspect, the present invention 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 LeuPro Pro X1 Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly Ser Asp Gly LysThr Tyr X8 Asn Asp Cys Phe Phe Cys Ser Lys Val Lys Lys Thr Asp Gly Thr LeuLys Phe Val His Phe Gly Lys Cys (SEQ ID NO: 3), wherein X1-X8 are as defined herein.

[0008] In some embodiments, the at least one mutant SPINK9 domain comprises an amino acid sequence X9 represented by Formula III 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 ProPro X1Gln X2 X3 X4 Cys X5 X6 17 As defined herein.

[0009] In some embodiments, the at least one mutant SPINK9 domain comprises an amino acid sequence represented by Formula IV: X9 X 10 X 11 Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys Lys LeuPro Pro X1 Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly Ser Asp Gly LysThr Tyr NO:136), where X1-X 11 As defined herein.

[0010] Preferably, the amino acid sequence represented by Formula I, Formula III and Formula IV is not the wild-type sequence of the SPINK9 domain (ie, SEQ ID NO: 2).

[0011] In other aspects, the present invention provides compositions comprising SPINK9 polypeptides, including pharmaceutical compositions; gene constructs comprising nucleic acid sequences encoding SPINK9 polypeptides; host cells comprising such gene constructs; and methods of using the SPINK9 polypeptides and their pharmaceutical compositions to treat diseases and conditions. Such SPINK9 polypeptides can be used to inhibit one or more KLKs, such as KLK5. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The average body weights of animals administered representative mutant SPINK9 polypeptides are shown.

[0013] Figure 2 Plasma concentrations of representative mutant SPINK9 polypeptides using the Fc+Fc ELISA method are shown.

[0014] Figure 3 Plasma concentrations of representative mutant SPINK9 polypeptides using the KLK5+Fc ELISA method are shown. DETAILED DESCRIPTION

[0015] KLK and its specific inhibitors play a key role in regulating proteolytic activity, especially in the skin. Changes in this balance that lead to dysregulation of KLK expression and / or activity are involved in a variety of human diseases and conditions (Paliouras, M, Biol Chem, 2006, Vol. 387, pp. 643-652). Specifically, serine protease KLK5 and serine protease inhibitor SPINK5 have each been shown to be involved in the regulation of epidermal desquamation. The SPINK5 gene encodes a 15-domain protein serine protease inhibitor Kazal type 5 (SPINK5; also known as lymphoepithelial Kazal-type associated inhibitor LEKTI).

[0016] SPINK5 is synthesized as three different high molecular weight precursors containing up to 15 inhibitory domains arranged as Kazal-like domains. The precursors are rapidly processed into shorter fragments and secreted extracellularly. Several 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 KLK inhibition lost at acidic pH similar to the outer layer of the skin. Loss-of-function mutations in SPINK5 result in the absence of KLK5 inhibitory activity and KLK5 hyperactivation.

[0017] Loss-of-function mutations in SPINK5 and the resulting increased activity of serine proteases (e.g., KLK5) have been shown to be causative factors in a variety of diseases, such as Netherton's syndrome and autosomal recessive ichthyosis with hypotrichosis. For example, Netherton's syndrome is associated with mutations in the SPINK5 gene, indicating that serine proteases are in fact important for skin physiology (Chavanas et al. 2000). Netherton's syndrome is characterized by congenital erythrodermic ichthyosis, hair defects, and atopic expression (Comel, 1949; Netherton, 1958; Chavanas et al., 2000). Severe complications are also associated with Netherton's syndrome, including recurrent bacterial infections, hypernatremic dehydration, and stunted growth.

[0018] 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 hyperkeratotic, such as the upper palmoplantar epidermis, and in clavi (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, two areas of the body that are not affected by the congenital SPINK5 mutation in Netherton syndrome are areas where SPINK9 is expressed, namely the soles of the feet and the palms of the hands. Thus, SPINK9 also plays a role in regulating skin physiology.

[0019] As disclosed herein, SPINK9 polypeptides and modifications and variants thereof can be utilized for their interaction with KLK proteases (e.g., KLK5). Therefore, provided herein are polypeptides and compositions thereof that target enzymes associated with diseases (especially skin disorders) (e.g., proteases of the KLK family), and methods of treatment using such polypeptides and compositions thereof. Such polypeptides and compositions thereof can be used to treat or prevent diseases and conditions characterized by abnormal KLK activity (e.g., abnormal KLK5 activity).

[0020] definition

[0021] 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. In general, the nomenclature used in conjunction with the 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 and their techniques are well known and commonly used in the art.

[0022] Unless otherwise indicated, the methods and techniques of the present invention 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 the specification. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press (1995); Lodish et al., "Molecular Cell Biology, 4th Edition", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th Edition", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th Edition", Sinauer Associates, Sunderland, MA (2000).

[0023] Unless otherwise defined herein, chemical terms used herein are used according to conventional usage in the art as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker, S., ed., McGraw-Hill, San Francisco, CA (1985).

[0024] All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, waivers or disclaimers of subject matter, and to the extent the incorporated material is inconsistent with the language disclosed herein, in which case the language of the present disclosure controls.

[0025] As used herein, the terms "administering," "administer," or "administration of" refer to the introduction of a medicament, including a therapeutic agent, into a subject and can be performed using one of a variety of methods known to those skilled in the art. For example, administration can be performed, for example, intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinal, intracerebrally, topically, and transdermally (by absorption). Administration can also be performed by refillable or biodegradable polymeric devices or other devices (e.g., patches and pumps) or formulations that provide for extended, slowed, or controlled release of a compound or agent. Administration can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0026] 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 invention, a "biologically active fragment" of SPINK9 is a fragment of SPINK9, including a mutant SPINK9 domain, which is capable of inhibiting or suppressing the activity of 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, including a mutant SPINK9 domain, which is capable of inhibiting or suppressing the activity of 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 a sequence of SEQ ID NO: 1 or 2).

[0027] As used herein, the phrase "conjoint administration" or "conjointly administered" refers to any form of administration of two or more different agents such that the second agent is administered while the previously administered agent is still effective in vivo (e.g., the two agents are simultaneously effective in the patient's body, which may include a synergistic effect of the two agents). For example, the different agents may be administered simultaneously or sequentially in the same formulation or in different formulations. Thus, patients receiving such treatments may benefit from the combined effects of the different agents.

[0028] As used herein, the phrase "consisting essentially of" when used to refer to compounds and compositions means that certain other components may be present in such compounds or compositions, i.e., those other components that do not substantially affect the basic characteristics of the compound or composition (e.g., the inhibitory activity of a SPINK9 polypeptide or the inhibitory activity of a mutant SPINK9 domain).

[0029] As used herein, the term "inhibit" means a reduction in an objectively measurable amount or degree. In various embodiments, "inhibit" means a reduction of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to a relevant control. In one embodiment, "inhibit" means a 100% reduction, i.e., cessation or elimination.

[0030] As used herein, the term "mutation" refers broadly to a polypeptide comprising 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 mutant polypeptide comprises an artificially introduced amino acid sequence change (e.g., an amino acid sequence change produced by human intervention in a laboratory or other facility). Such mutant polypeptides may contain a single change or multiple changes in the amino acid sequence compared to a reference polypeptide. Such amino acid changes include, but are not limited to, substitutions, insertions, and deletions. Guidance for substitutions, insertions, or deletions may be based on the comparison of amino acid sequences of different variant proteins or proteins from different species.

[0031] As used herein, the term "mutant SPINK9 domain" refers to a sequence in a polypeptide comprising 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. Compared to wild-type SPINK9, such "mutant SPINK9 domains" may contain a single change in amino acid sequence or multiple changes in amino acid sequence. 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.

[0032] As used herein, the terms "patient", "subject" or "individual" are used interchangeably herein and refer to 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 cattle, pigs, etc.), companion animals (such as dogs, cats, etc.), and rodents (such as mice and rats). In one embodiment, the individual is a human.

[0033] The phrase "pharmaceutically acceptable" is recognized in the art. 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 humans and animals without excessive toxicity, irritation, allergic response or other problems or complications that match a reasonable benefit / risk ratio within the scope of sound medical judgment.

[0034] 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 use in or compatible with the treatment of a patient.

[0035] The terms "preventing" or "prevention" are art-recognized and well understood in the art when used in relation to a disease (e.g., Netherton's syndrome) or any other medical condition, and include administering a composition that reduces the frequency of symptoms of, or delays the onset of, a medical condition in an individual relative to an individual not receiving the composition. Thus, preventing Netherton's syndrome includes, for example, reducing the number or severity of atopic expressions of the disease in a population of individuals receiving prophylactic treatment relative to an untreated control population, and / or delaying the onset of atopic expressions of the disease in a population of individuals receiving prophylactic treatment relative to an untreated control population, e.g., the reduction / delay is by a statistically and / or clinically significant amount.

[0036] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host to form an active compound (e.g., a compound of the present invention) after administration. Typical examples of prodrugs include compounds having biologically unstable or cleavable (protecting) groups on the functional portion of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminized, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramidates as biologically unstable or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference in their entirety. The prodrugs of the present invention are metabolized to produce SPINK9 polypeptides. The present invention 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.

[0037] 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 that can be used to formulate the SPINK9 polypeptides 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: (1) water, any and all solvents, dispersion media, diluents or other liquid vehicles; (2) dispersion or suspension aids; (3) surfactants and surface active agents; (4) isotonic, thickening or emulsifying agents; (5) preservatives, antioxidants, chelating agents, solid binders, lubricants; (6) sugars, such as polydextrose, lactose, glucose and sucrose; (7) starches, such as corn starch and potato starch; (8) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (9) 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 glycerol, 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, phosphate buffer solution; (14) osmotic pressure regulators; (15) stabilizers and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various carriers for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional carrier is incompatible with the substance or its derivatives, for example by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other components of the composition, its use is contemplated to be within the scope of the present invention.

[0038] As used herein, the term "sequence identity" refers to the sequence similarity between two peptide / polypeptide / protein molecules. For example, when one position in both of the two compared sequences is occupied by the same amino acid monomer subunit, the molecules are consistent at the position. Without being bound by a particular theory or methodology, the percentage of sequence identity between two sequences can be considered as a function of the number of consistent positions shared by the two sequences divided by the number of compared positions and multiplied by 100. For example, if 6 of the 10 positions in the two sequences are the same, the two sequences have 60% sequence identity. In general, the two sequences are compared when they are aligned to obtain the most consistent positions.

[0039] As used herein, the term "SPINK9 polypeptide" refers to a polypeptide comprising a mutant SPINK9 domain and an optional additional domain (e.g., an Fc portion). In certain embodiments, a "SPINK9 polypeptide" comprises more than one mutant SPINK9 domain (e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 2 mutant SPINK9 domains) and an optional additional domain (e.g., an Fc portion). In some embodiments, the SPINK9 polypeptide may comprise a plurality of bound polypeptide chains (e.g., in the form of polypeptide dimers connected via an Fc portion or other dimerization domains). In some embodiments, each polypeptide chain in the SPINK9 polypeptide comprises a mutant SPINK9 domain.

[0040] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or pharmaceutical agent is an amount of the drug or pharmaceutical agent that will have the intended therapeutic effect when administered to an individual. The full therapeutic effect may not occur when a single dose is administered, and may occur only after a series of doses are administered. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for an individual will depend, for example, on the individual's size, health, and age, as well as the nature and extent of the condition being treated. One skilled in the art can readily determine the effective amount for a given situation by routine experimentation.

[0041] As is well understood in the art, the terms "treat," "treating," and "treatment" refer to taking steps to obtain beneficial or desired results, including clinical results. Favorable or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, lessening of the extent of the disease, stabilization (i.e., not worsening) of the disease state, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival as compared to expected survival if not receiving treatment.

[0042] Amino acid residues can be referred to by their corresponding three letter codes or their corresponding single letter codes. Various three letter and single letter codes for 20 commonly used amino acids are provided below.

[0043] Amino Acids 3 letter code 1 letter code Amino Acids 3 letter code 1 letter code Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R Glutamine Glu Q Aspartic acid Asn N Glutamate Glu E Aspartic acid Asp D Serine Ser S Threonine Thr T

[0044] Overview

[0045] The present invention provides SPINK9 polypeptides comprising (e.g., at least one) mutant SPINK9 domain. The SPINK9 polypeptide inhibits or otherwise weakens the active KLK via the interaction of the mutant SPINK9 domain with the active KLK. The active KLK may 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.

[0046] In some embodiments, the SPINK9 polypeptide is a selective inhibitor of 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), and the 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., at least 20% or greater, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or greater) than the activity of another KLK family member or other serine protease.

[0047] 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.

[0048] In a preferred embodiment, the SPINK9 polypeptide inhibits KLK5. In another preferred embodiment, the SPINK9 polypeptide is a selective inhibitor of KLK5.

[0049] Mutation of SPINK9 domain

[0050] The mutant SPINK9 domain may comprise all or part of the wild-type SPINK9 sequence of SEQ ID NO: 1 (which includes the signal peptide) or all or part of the wild-type SPINK9 sequence of SEQ ID NO: 2 (which does not include the signal sequence). In a preferred embodiment, the mutant SPINK9 domain comprises all or part of the wild-type SPINK9 of SEQ ID NO: 2.

[0051] MRATAIVLLLALTLATMFSIECAKQTKQMVDCSHYKKLPPGQQRFCHHMYDPICGSDGKTYKNDCFFCSKVKKTDGTLKFVHFGKC(SEQ ID NO:1)

[0052] IECAKQTKQMVDCSHYKKLPPGQQRFCHHMYDPICGSDGKTYKNDCFFCSKVKKTDG TLKFVHFGKC(SEQ ID NO:2)

[0053] In some embodiments, the mutant SPINK9 domain or a 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 with SEQ ID NO: 1 or 2. In certain embodiments, the mutant SPINK9 domain or a biologically active fragment thereof shares 70% or greater (i.e., 75%, 80%, 85%, 90% or 95% or greater) sequence identity with SEQ ID NO: 1 or 2. In a preferred embodiment, the mutant SPINK9 domain or its biologically active fragment shares 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2. In another preferred embodiment, the mutant SPINK9 domain or its biologically active fragment shares 70% or greater (i.e., 75%, 80%, 85%, 90% or 95% or greater) sequence identity with SEQ ID NO: 2.

[0054] The mutant SPINK9 domain may comprise one or more amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the mutant SPINK9 domain comprises 1-20, 1-15, 1-10, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2 or 1 amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the mutant SPINK9 domain comprises 2-7, 3-7, 4-7, 5-7, 6-7 or 7 amino acid changes relative to SEQ ID NO: 1 or 2. In certain embodiments, the mutant SPINK9 domain comprises 2-15 (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 mutant SPINK9 domain comprises more than 1 and less than 20 (e.g., 2-7 or 2-15) amino acid changes relative to SEQ ID NO: 1 or 2.

[0055] In some embodiments, at least one of the one or more amino acid changes in the mutant SPINK9 domain occurs at or in amino acid positions 40 to 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 in amino acid positions 21-34 of SEQ ID NO: 2. The referenced amino acid positions contain the reactive P1 / P1' active site of SPINK9.

[0056] 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 in 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 in positions 40 to 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 in 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 in 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 in positions 21-34 of SEQ ID NO: 2.

[0057] 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 in 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 in positions 40 to 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 in 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 in 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 in 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 in positions 21-34 of SEQ ID NO: 2.

[0058] 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 in amino acid positions 40 to 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 in amino acid positions 21-34 of SEQ ID NO: 2.

[0059] In one embodiment, the mutant SPINK9 domain comprises a sequence that shares 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid positions 1-20 and 35-67 of SEQ ID NO: 2, and contains at least one amino acid change (e.g., amino acid substitution) at positions 21-34 of SEQ ID NO: 2. In a preferred embodiment, the sequence identity with 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%).

[0060] 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 shown in SEQ ID NO:2; wherein the mutant SPINK9 domain further comprises at least one amino acid substitution at position 21-34 of SEQ ID NO:2.

[0061] In one embodiment, the mutant SPINK9 polypeptide comprises, in N-terminal to C-terminal order, a first amino acid sequence corresponding to amino acids 1-20 of SEQ ID NO:2, a second amino acid sequence corresponding to amino acids 21-34 of SEQ ID NO:2, and a third amino acid sequence corresponding to amino acids 35-67 of SEQ ID NO:2.

[0062] In a first aspect of such embodiments: (i) the first amino acid sequence shares at least 70% sequence identity with amino acids 1-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid substitution relative to amino acids 21-34 of SEQ ID NO:2; and (iii) the 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.

[0063] In a second aspect of such embodiments: (i) the first amino acid sequence optionally contains the 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., amino acid substitution) relative 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0064] In a third aspect of such embodiments: (i) the first amino acid sequence optionally contains the 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., amino acid substitution) at position 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0065] 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 with amino acids 10-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0066] 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 with amino acids 4-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0067] In a sixth aspect of such embodiments: (i) the first amino acid sequence comprises a deletion of at least 1, 2 or 3 amino acids at position 1, 2 or 3 of SEQ ID NO:2 and optionally shares at least 70% sequence identity with amino acids 4-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0068] In a seventh aspect of such embodiments: (i) the first amino acid sequence comprises a deletion of the amino acids at positions 1, 2 and 3 of SEQ ID NO:2 and optionally shares at least 70% sequence identity with amino acids 4-20 of SEQ ID NO:2; (ii) the second amino acid sequence comprises at least one amino acid change (e.g., amino acid substitution) at positions 21-34 of SEQ ID NO:2; and (iii) the 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 with SEQ ID NO:2 and optionally comprises an amino acid change (i.e., amino acid substitution) at position 43.

[0069] In any one of the first to seventh aspects above, 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 21-34 of SEQ ID NO:2.

[0070] 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 (eg, amino acid substitutions) at positions 21-34 of SEQ ID NO:2.

[0071] 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 comprises 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.

[0072] In any one of the first to seventh aspects above, the second amino acid sequence comprises 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 may contain amino acid changes at only one position (e.g., position 28), at only two positions (e.g., positions 28 and 29), at only three positions (e.g., positions 28, 29 and 30), at only four positions (e.g., positions 25, 28, 29 and 30), at only five positions (e.g., positions 25, 26, 28, 29 and 30), at only six positions (e.g., positions 22, 25, 26, 28, 29 and 30), or at all seven positions (e.g., positions 22, 24, 25, 26, 28, 29 and 30).

[0073] 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: (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; (iii) the amino acid at position 25 is selected from The amino acid at position 26 is selected from the group consisting of Phe, Tyr, Trp, Met, His, Lys or Asn; (v) the amino acid at position 28 is selected from the group consisting of His, Thr, Ser or Gly; (vi) the amino acid at position 29 is selected from the group consisting of His, Arg or Lys; (vii) the amino acid at position 30 is selected from the group consisting of Met, Glu or Asp; or (viii) any combination thereof.

[0074] 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 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 at position 22 relative to 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 at position 24 relative to SEQ ID NO: 2, or the amino acid at position 24 of SEQ ID NO: 2 is Gln; (iii) comprises a substitution of Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys or His amino acid at position 22 relative to SEQ ID NO: 2, or the amino acid at position 22 of SEQ ID NO: 2 is Gly; The amino acid at position 25 of SEQ ID NO:2 comprises a substitution of 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 relative to SEQ ID NO:2 comprises a substitution of 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 relative to SEQ ID NO:2 comprises a substitution of Thr, Ser or Gly, or the amino acid at position 28 of SEQ ID NO:2 is His; (vi) the amino acid at position 29 relative to SEQ ID NO:2 comprises a substitution of Arg or Lys, or the amino acid at position 29 of SEQ ID NO:2 is His; (vii) the amino acid at position 30 relative to SEQ ID NO:2 comprises a substitution of Glu or Asp, or the amino acid at position 31 of SEQ ID NO:2 is The amino acid at position 30 of NO: 2 is Met; or (viii) any combination thereof.

[0075] 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: (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; (iii) the amino acid at position 25 is selected from 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 28-30 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.

[0076] 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 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 at position 22 relative to 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 at position 24 relative to SEQ ID NO: 2, or the amino acid at position 24 of SEQ ID NO: 2 is Gln; (iii) comprises a substitution of Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys or His amino acid at position 22 relative to SEQ ID NO: 2, or the amino acid at position 22 of SEQ ID NO: 2 is Gly; The amino acid at position 25 of SEQ ID NO:2 comprises a substitution of 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 relative to SEQ ID NO:2 comprises a substitution of Tyr, Trp, Met, His, Lys or Asn, or the amino acid at position 26 of SEQ ID NO:2 is Phe; (v) the amino acids at positions 28-30 relative to SEQ ID NO:2 comprise a substitution of Thr-Arg-Glu; Thr-Arg-Asp; Thr-Lys-Glu; Thr-Lys-Asp; Ser-Arg-Glu; or Gly-Arg-Asp; or (vi) any combination thereof.

[0077] 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: (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.

[0078] 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 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 at the amino acid at position 22 relative to 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 at the amino acid at position 24 relative to 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 at the amino acid at position 25 relative to SEQ ID NO: 2, or the amino acid at position 25 of SEQ ID NO: 2 is Arg; (iv) comprises a substitution of a Tyr or Trp amino acid at the amino acid at position 26 relative to SEQ ID NO: 2, or the amino acid at position 26 of SEQ ID NO: 2 is Phe; (v) comprises a substitution of a Tyr or Trp amino acid at the amino acid at position 26 relative to SEQ ID NO: 2, or the amino acid at position 26 of SEQ ID NO: 2 is Phe; The amino acid at position 28 of SEQ ID NO:2 comprises a substitution of 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 relative to SEQ ID NO:2 comprises a substitution of Arg or Lys amino acid, or the amino acid at position 29 of SEQ ID NO:2 is His; (vii) the amino acid at position 30 relative to SEQ ID NO:2 comprises a substitution of Glu or Asp amino acid, or the amino acid at position 30 of SEQ ID NO:2 is Met; or (viii) any combination thereof.

[0079] 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: (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.

[0080] 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 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 at the amino acid at position 22 relative to 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 at the amino acid at position 24 relative to 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 at the amino acid at position 25 relative to SEQ ID NO: 2, or the amino acid at position 25 of SEQ ID NO: 2 is Arg; (iv) comprises a substitution of a Tyr or Trp amino acid at the amino acid at position 26 relative to SEQ ID NO: 2, or the amino acid at position 26 of SEQ ID NO: 2 is Phe; (v) comprises a substitution of a Tyr or Trp amino acid at the amino acid at position 26 relative to SEQ ID NO: 2, or the amino acid at position 26 of SEQ ID NO: 2 is Phe; The amino acids at positions 28-30 of NO:2 comprise substitutions of Thr-Arg-Glu; Thr-Arg-Asp; Thr-Lys-Glu; Thr-Lys-Asp; Ser-Arg-Glu; or Gly-Arg-Asp amino acids; or (vi) any combination thereof.

[0081] In any one of the first to seventh aspects above, the amino acid change optionally present at position 43 of the third amino acid sequence is an amino acid substitution. In any one of the first to seventh aspects above, the third amino acid sequence comprises a substitution of a Gly amino acid for the amino acid at position 43 relative to SEQ ID NO: 2.

[0082] In any one of the first to seventh aspects above, at least one of the amino acid changes in SEQ ID NO: 2 is an amino acid substitution. In any one of the first to seventh aspects above, the amino acid changes in SEQ ID NO: 2 are amino acid substitutions.

[0083] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of an 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).

[0084] Ile Glu Cys Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr LysLys Leu Pro Pro X1Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly Ser Asp GlyLys Thr Tyr Cys (Formula I; SEQ ID NO:3).

[0085] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of an 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).

[0086] X9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 Met Val Asp Cys Ser His Tyr Lys Lys LeuPro Pro X1 Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly SerAsp Gly Lys ThrTyr

[0087] In some embodiments, the mutant SPINK9 domain comprises, consists of, or consists essentially of an 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).

[0088] X9 X 10 X 11Ala Lys Gln Thr Lys Gln Met Val Asp Cys Ser His Tyr Lys LysLeu Pro Pro X1 Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly SerAsp Gly LysThr Tyr Cys (Formula I; SEQ ID NO: 136).

[0089] In a preferred 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 X 1-X17 As indicated in Table 1 or Table 2 herein.

[0090] Table 1 - X of Formula I, Formula III or Formula IV 1-X17 Representative amino acid substitutions

[0091]

[0092]

[0093] Table 2 - X1-X7 and X of Formula I, Formula III or Formula IV 19 -X 27 Representative amino acid substitutions

[0094]

[0095]

[0096] 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 As indicated in Table 3 or Table 4 herein.

[0097] Table 3 - X of Formula I, Formula III or Formula IV 1-X17 Representative amino acid substitutions

[0098]

[0099]

[0100] Table 4 - X of Formula I, Formula III or Formula IV 1-X17 and X19 -X 27 Representative amino acid substitutions

[0101]

[0102] 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 as shown in Table 5.

[0103] Table 5: Exemplary mutant SPINK9 domains

[0104]

[0105]

[0106]

[0107] In some embodiments, the mutant SPINK9 domain has a sequence disclosed herein (e.g., SEQ ID NOs: 4-23 and 115-134), further comprising 1 or more (e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 8, or 1 to 5) conservative sequence modifications, wherein the one or more conservative sequence modifications are not at an amino acid corresponding to amino acid position 41, 43, 44, 45, 47, 48, or 49 of SEQ ID NO: 1 or position 22, 24, 25, 26, 28, 29, or 30 of SEQ ID NO: 2. In some embodiments, the mutant SPINK9 domain has a sequence disclosed herein, further comprising 1 or more (e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 8, or 1 to 5) conservative sequence modifications, wherein the one or more conservative sequence modifications are not at an amino acid corresponding to amino acid position 32, 41, 43, 44, 45, 46, 47, 48, 49, 54, 65, 68, or 86 of SEQ ID NO: 1 or position 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 amino acid modifications that do not significantly affect or alter the interaction of the mutant SPINK9 domain with a homologous peptide (e.g., a KLK protease, e.g., KLK5), and optionally includes natural variations in the amino acid sequence of SPINK9. Such conservative substitutions include, but are not limited to, amino acid substitutions, additions (e.g., addition of amino acids to the N-terminus or C-terminus of a peptide) and deletions (e.g., deletion of amino acids from the N-terminus or C-terminus of a peptide). In one embodiment, a conservative amino acid modification is a modification in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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 may be replaced by other amino acid residues from the same side chain family, and the variant peptides may 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.

[0108] SPINK9 peptide

[0109] The present invention 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 can be provided as a pharmaceutically acceptable salt. Any mutant SPINK9 domain described herein can be used in a SPINK9 polypeptide.

[0110] 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 specific cell types, providing delayed or controlled release in vivo, or any combination of the foregoing. Representative additional domains are discussed herein.

[0111] In some embodiments, the SPINK9 polypeptide comprises a single mutant SPINK9 domain, wherein the single mutant SPINK9 domain 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 the amino acid sequence set forth in Tables 1 to 5.

[0112] In some embodiments, the SPINK9 polypeptide comprises two to twenty (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) independently selected mutant SPINK9 domains, wherein each mutant SPINK9 domain 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 the amino acid sequence set forth in Tables 1 to 5.

[0113] In some embodiments, the SPINK9 polypeptide comprises a first and a second independently selected mutant SPINK9 domain; and each of the first and second mutant SPINK9 domains comprises an independently selected amino acid sequence selected from a mutant SPINK9 domain amino acid sequence as disclosed herein, including but not limited to the amino acid sequence of Formula I, III or IV and the amino acid sequence set forth in Tables 1 to 5. In some such embodiments, the first and second mutant SPINK9 domains are the same. In some such embodiments, the first and second mutant SPINK9 domains are not the same. In a preferred embodiment, each mutant SPINK9 domain is independently selected from Table 5.

[0114] In some embodiments, the SPINK9 polypeptide comprises a first, second, and third independently selected mutant SPINK9 domain; and each of the first, second, and third mutant SPINK9 domains comprises an independently selected amino acid sequence selected from a mutant SPINK9 domain amino acid sequence as disclosed herein, including but not limited to the amino acid sequence of Formula I, III, or IV and the amino acid sequence set forth in Tables 1 to 5. In some such embodiments, two and only two of the mutant SPINK9 domains are identical. In some such embodiments, the first, second, and third mutant SPINK9 domains are identical. In some such embodiments, the first, second, and third mutant SPINK9 domains are not identical. In a preferred embodiment, each mutant SPINK9 domain is independently selected from Table 5.

[0115] When the SPINK9 polypeptide comprises more than one (e.g., 2 to 20) mutant SPINK9 domains, the mutant SPINK9 domains may be positioned adjacent to each other in the SPINK9 polypeptide, or the additional domain may be positioned between one or more mutant SPINK9 domains. In some embodiments, all mutant SPINK9 domains in the SPINK9 polypeptide are positioned adjacent to each other.

[0116] In some embodiments, when there are 2 or more adjacent mutant SPINK9 domains, the mutant SPINK9 domains are connected by direct fusion (e.g., there is no intervening amino acid sequence between adjacent mutant SPINK9 domains). In some embodiments, when there are 2 or more adjacent mutant SPINK9 domains, the mutant SPINK9 domains are connected by a linker sequence. In some embodiments, when there are more than 2 mutant SPINK9 domains, the mutant SPINK9 domains are connected by a combination of direct fusion and linker sequences. In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is connected to an adjacent mutant SPINK9 domain by a linker sequence. In other embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is connected to an adjacent mutant SPINK9 domain by direct fusion.

[0117] In some embodiments, the SPINK9 polypeptide comprises a plurality of related polypeptide chains (e.g., polypeptide dimers connected via an Fc portion or other dimerization domain), wherein at least one polypeptide chain in the SPINK9 polypeptide comprises at least one (e.g., 2 to 20) mutant SPINK9 domains.

[0118] In some embodiments, the SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion or other dimerization domain, wherein at least one of the first and second polypeptide chains comprises at least one (e.g., 2 to 20) mutant SPINK9 domains. In some such embodiments, the mutant SPINK9 domains are independently selected from any mutant SPINK9 domains disclosed herein, including but not limited to mutant SPINK9 domains comprising the amino acid sequence of Formula I, III or IV or the amino acid sequence set forth in Tables 1 to 5. In a preferred embodiment, the first and second mutant SPINK9 domains are independently selected from Table 5.

[0119] In some embodiments, the SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via 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 domain is independently selected from any mutant SPINK9 domain disclosed herein, including but not limited to a mutant SPINK9 domain comprising an amino acid sequence of Formula I, III or IV or an amino acid sequence described in Tables 1 to 5. In some such embodiments, the first and second mutant SPINK9 domains are the same. In some such embodiments, the first and second mutant SPINK9 domains are not the same. In a preferred embodiment, the first and second mutant SPINK9 domains are independently selected from Table 5.

[0120] In some embodiments, the SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via 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 domain is independently selected from any mutant SPINK9 domain disclosed herein, including but not limited to a mutant SPINK9 domain comprising an amino acid sequence of Formula I, III or IV or an amino acid sequence described in Tables 1 to 5. In some such embodiments, at least two of the first to fourth mutant SPINK9 domains are the same. In some such embodiments, each of the first to fourth mutant SPINK9 domains is the same. In some such embodiments, each of the first to fourth mutant SPINK9 domains is not the same. In a preferred embodiment, the first to fourth mutant SPINK9 domains are independently selected from Table 5.

[0121] In some embodiments, the SPINK9 polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion or other dimerization domain, wherein the first polypeptide chain comprises the first, second and third mutant SPINK9 domains and the second polypeptide chain comprises the 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 the mutant SPINK9 domains comprising the amino acid sequence of Formula I, III or IV or the amino acid sequence described in Tables 1 to 5. In some such embodiments, at least two of the first to sixth mutant SPINK9 domains are the same. In some such embodiments, each of the first to sixth mutant SPINK9 domains is the same. In some such embodiments, each of the first to sixth mutant SPINK9 domains is not the same. In a preferred embodiment, the first to sixth mutant SPINK9 domains are independently selected from Table 5.

[0122] When the SPINK9 polypeptide comprises a first and a second polypeptide chain and the first and / or the second polypeptide chain comprises more than one (e.g., 2 to 20) mutant SPINK9 domains, the mutant SPINK9 domains in the first and / or the second polypeptide chain may be positioned adjacent to each other in the first and / or the second polypeptide chain, or the additional domain may be positioned between one or more mutant SPINK9 domains in the first and / or the second polypeptide chain. In some embodiments, all mutant SPINK9 domains in the first and / or the second polypeptide chain are positioned adjacent to each other.

[0123] In some embodiments, when 2 or more adjacent mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are connected by direct fusion (e.g., there is no intervening amino acid sequence between adjacent mutant SPINK9 domains). In some embodiments, when 2 or more adjacent mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are connected by a linker sequence. In some embodiments, when more than 2 mutant SPINK9 domains are present on the first and / or second polypeptide chains, the mutant SPINK9 domains are connected by a combination of direct fusion and linker sequences. In some embodiments, at least one mutant SPINK9 domain of the first and / or second polypeptide chain is connected to an adjacent mutant SPINK9 domain by a linker sequence. In other embodiments, at least one mutant SPINK9 domain of the first and / or second polypeptide chain is connected to an adjacent mutant SPINK9 domain by direct fusion.

[0124] The SPINK9 polypeptide may further comprise an additional domain. In some embodiments, the SPINK9 polypeptide comprises 1 to 5 additional domains. Preferably, in addition to inhibiting active pancreatic vasodilator-related peptidases (eg, KLK5), other domains provide additional functions for the SPINK9 polypeptide.

[0125] 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) in endothelial cells of blood vessels, and after binding, the Fc region containing the polypeptide is protected from degradation and released back into the circulation, thereby increasing the half-life of the attached polypeptide after administration to an individual. The Fc region can be linked to the SPINK9 polypeptide to optimize the pharmacokinetic and pharmacodynamic properties of the polypeptide.

[0126] The term "Fc region" means the C-terminal region of the heavy chain of an immunoglobulin, which can be produced by papain digestion of an intact antibody. The Fc region may be a natural amino acid sequence or may contain one or more modifications. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. It is known in the art that the amino acid sequence of the Fc portion is altered to change the antibody effector function (e.g., U.S. Patent 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), and increases half-life and reduces the clearance rate of polypeptides. In some cases, these effector functions are desirable for therapeutic immunoglobulins, but in other cases, they may be unnecessary or even harmful depending on the therapeutic goal.

[0127] In some embodiments, the Fc region or its functional fragment is connected to the SPINK9 polypeptide by direct fusion or through a linker sequence. In some embodiments, the Fc region or its functional fragment can be connected to the SPINK9 polypeptide at the N-terminus or C-terminus of the mutant SPINK9 domain.

[0128] In some embodiments, the immunoglobulin Fc region is of human origin. In some embodiments, the immunoglobulin Fc region is from or derived from non-human animals, including but not limited to rodents, such as but not limited to mice and rats; mammals, such as but not limited to rabbits, cattle, pigs, dogs, cynomolgus macaques, marmosets, rhesus monkeys and birds, such as but not limited to chickens. Preferably, the immunoglobulin Fc region is of human origin or derived from non-human animals and is known as "humanization" 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.

[0129] 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 may comprise an amino acid insertion, deletion, substitution or chemical modification. For example, the Fc region modification may be performed to increase or decrease complement fixation, increase or decrease ADCC or CDC, increase or decrease binding to FcR, increase or decrease binding to FcRn, modify glycosylation, or any combination thereof. Various Fc modifications are known in the art and have been 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 number 1296). Any Fc modification known in the art can be applied to a SPINK9 polypeptide comprising an Fc region disclosed herein. In some embodiments, the Fc modification is an amino acid substitution.

[0130] In some embodiments, the SPINK9 polypeptide comprising a modified Fc region is characterized by reduced binding (e.g., minimal or absent binding) to a human Fc receptor (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or a combination thereof). In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduced Fc receptor binding compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduced ADCC compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, decreased ADCC is achieved by the 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).

[0131] In some embodiments, the SPINK9 polypeptide comprising a modified Fc region is characterized by reduced binding (e.g., minimal or absent binding) to complement proteins compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduced C1q binding compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduced CDC compared to a SPINK9 polypeptide comprising a wild-type Fc region.

[0132] In some embodiments, the SPINK9 polypeptide comprising a modified Fc region is characterized by increased affinity for human neonatal Fc receptor (FcRn) at low pH (e.g., pH 5.8 to 6.0) compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) affinity for FcRn at low pH compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, increased affinity for human neonatal Fc receptor (FcRn) at low pH is achieved by 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).

[0133] In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits minimal or reduced glycosylation compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, the SPINK9 polypeptide comprising a modified Fc region exhibits at least 5% or more (e.g., 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) reduced glycosylation compared to a SPINK9 polypeptide comprising a wild-type Fc region. In some embodiments, minimal or reduced glycosylation is achieved by amino acid substitution N297X, wherein X is any amino acid other than N (e.g., N297A substitution) (numbering according to EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969).

[0134] In some embodiments, the Fc region is from an IgG1 antibody, in particular a human IgG1 antibody. In some embodiments, the modified Fc region is from an IgG1 antibody, in particular a human IgG1 antibody, and comprises one or more of the 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, wherein X is any amino acid other than N (e.g., N297A substitution), P228S A330S, P331S, G237A, E233P / L234V / L235A, A327G / A330S / P331S or L234F / L235E / P331S (numbered according to EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969). In some embodiments, one or more additional mutations are included in such 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.

[0135] In some embodiments, the Fc region comprises, consists of, or consists essentially of the amino acid sequence of Formula II (SEQ ID NO: 24):

[0136] Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro X1 Cys Pro Ala Pro X2X3 X4 Gly X5 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu X6 Ile X7Arg X8 Pro Glu Val Thr Cys Val Val GluGlu Gln Tyr 10 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val X 11 His X12 Asp TrpLeu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys X 13 Leu Pro X 14 X 15 Ile GluLys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu ProPro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser LysLeu 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;

[0137] in

[0138] X1 is P or S;

[0139] X2 is E or P;

[0140] X3 and X4 are independently L or A;

[0141] X5 G or A;

[0142] X6 M or Y;

[0143] X7 S or T;

[0144] X8 T or E;

[0145] X9V or E;

[0146] X 10 for N or A;

[0147] X 11 for L or D;

[0148] X 12 for Q or H;

[0149] X 13 for A or G;

[0150] X 14 for A or S;

[0151] X 15 for P or S;

[0152] X 16 for M or L;

[0153] X 17 for H or K; and

[0154] X 18 for N , S or F; wherein the wild-type amino acid is underlined and the amino acid not underlined is a non-wild-type (substituted) amino acid.

[0155] In some embodiments of Formula II, X6-X7-X8 are MST, and X1 to X5 and X9 to X 18 In some embodiments of Formula II, X6-X7-X8 are YTE, and X1 to X5 and X9 to X 18 In some embodiments of Formula II, X6-X7-X8 are YTE, and X1 to X5 and X9 to X 18 Each of which is a wild-type amino acid.

[0156] In some embodiments of Formula II, X 16 and X 18 for M and N, and X1 to X 15 and X 17 At least one of X is a non-wild-type amino acid. 16 and X 18 for L and S, and X1 to X 15 and X 17In some embodiments of Formula II, X 16 and X 18 for L and S, and X1 to X 15 and X 17 Each of which is a wild-type amino acid.

[0157] In some embodiments of Formula II, X3-X4 are LL, and X1 to X2 and X5 to X 18 In some embodiments of Formula II, X3-X4 are AA, and X1 to X2 and X5 to X 18 In some embodiments of Formula II, X3-X4 are AA, and X1 to X2 and X5 to X 18 Each of which is a wild-type amino acid.

[0158] In some embodiments of Formula II, X9, X 11 and X 12 are V, L and Q, and X1 to X8, X 10 and X 13 To X 18 At least one of X9, X10, X20, X30, X40, X50, X60, X70, X80, X90, X100, X110, X120, X130, X140, X150, X160, X170, X180, X190, X211, X220, X311, X420, X53, X64, X71 11 and X 12 are E, D, and H, and X1 to X8, X 10 and X 13 To X 18 In some embodiments of Formula II, X9, X 11 and X 12 are E, D, and H, and X1 to X8, X 10 and X 13 To X 18 Each of which is a wild-type amino acid.

[0159] 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 To X 17 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 To X 17 In some embodiments of Formula II, X 11 , X12 and X 18 are D, H and S, and X1 to X 10 and X 13 To X 17 Each of which is a wild-type amino acid.

[0160] In some embodiments of Formula II, X 17 -X 18 is HN, and X1 to X 16 At least one of X is a non-wild-type amino acid. 17 -X 18 is KF, and X1 to X 16 In some embodiments of Formula II, X 17 -X 18 is KF, and X1 to X 16 Each of which is a wild-type amino acid.

[0161] In some embodiments of Formula II, X3-X4 is AA, X 16 and X 18 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 to X2, X5 and X9 to X1 are each a wild-type amino acid. 18 Each of which is a wild-type amino acid.

[0162] In some embodiments of Formula II, X 10 is N, and X1 to X9 and X 11 To X 18 At least one of X is a non-wild-type amino acid. 10 is A, and X1 to X9 and X 11 To X 18 In some embodiments of Formula II, X 10 is A, and X1 to X9 and X 11 To X 18 Each of which is a wild-type amino acid.

[0163] In some embodiments of Formula II, X 10 is A, and X3-X4 are AA, and X1 to X2, X5 to X9 and X 11 To X 18 At least one or all of them are wild-type amino acids.

[0164] In some embodiments of Formula II, X 10 is A, and X 16 and X 18 L and S, and X1 to X9, X 11 To X 15 and X 17 At least one or all of them are wild-type amino acids.

[0165] In some embodiments of Formula II, X 10 is A, and X6-X7-X8 are YTE, and X1 to X5, X9 and X 11 To X 18 At least one or all of them are wild-type amino acids.

[0166] In some embodiments of Formula II, X 10 is A, X3-X4 is AA, and X 16 and X 18 L and S, and X1 to X2, X5 to X9, X 11 To X 15 and X 17 At least one or all of them are wild-type amino acids.

[0167] In some embodiments of Formula II, X 10 A, X3-X4 are AA, and X6-X7-X8 are YTE, and X1 to X2, X5 and X9 to X 18 At least one or all of them are wild-type amino acids.

[0168] As used in the present invention, "linker" or "linker sequence" means an amino acid sequence of natural and / or synthetic origin. The linker sequence may comprise a linear amino acid chain, wherein 20 naturally occurring amino acids are monomer building blocks. The length of the linker may be 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 may comprise a repeating amino acid sequence or a naturally occurring polypeptide sequence. In some embodiments, the linker is used to ensure that the polypeptides or polypeptide domains connected to each other within the polypeptide can perform their biological activity by allowing the polypeptides or polypeptide domains to fold correctly and / or to be properly presented. The linker may 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, the linker sequence is rich in glycine, glutamine, and / or serine residues. These residues can be arranged in small repeating units and can be repeated as needed. In some embodiments, the linker comprises a single amino acid, which is repeated 10 to 20 times. In some embodiments, the linker comprises a cleavage site and the cleavage site is optionally positioned adjacent to the mutant SPINK9 domain (suitable cleavage sites are known in the art and described herein). By way of example, but not limitation, the SPINK9 polypeptides described herein may comprise 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), GGGGSGGGGSGGGGS (SEQ ID NO:32), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:33), and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:34).

[0169] In some embodiments, the linker sequence is encoded by a nucleic acid molecule and can therefore be expressed in a recombinant manner. In some embodiments, the linker sequence includes one or more bonds that are not peptide bonds (such alternative linkages are known in the art and described herein). In such embodiments, the SPINK9 polypeptide containing such a linker sequence can be produced by a non-recombinant method as described herein or by a combination of recombinant and non-recombinant methods.

[0170] In some embodiments, the linker sequence comprises a cleavage site. Such cleavage sites can be recognized by proteases naturally present in an individual, such as proprotein convertases (PPCs), more preferably subtilisin-like proprotein convertases (SPCs). SPCs are a family of calcium-dependent lyases that act on binary sites of 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); wherein↓ indicates a cleavage point. The cleavage site disclosed herein may be located at the N-terminus, C-terminus, or an 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 serves solely as a linker sequence.

[0171] In some embodiments, the SPINK9 polypeptide provided herein further comprises a signal sequence. In some such embodiments, the signal sequence is connected to the amino (N) end of the polypeptide by direct fusion or by a linker sequence. In other embodiments, the signal sequence can be connected to the carboxyl (C) end of the polypeptide by direct fusion or by a linker sequence.

[0172] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO: 39, or a sequence having 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:

[0173] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0174] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0175] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0176] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39).

[0177] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:40, or a sequence having 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:

[0178] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0179] FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA

[0180] PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0181] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40).

[0182] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:41, or a sequence having 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:

[0183] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0184] FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA

[0185] PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0186] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 41).

[0187] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:42, or a sequence having 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:

[0188] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKF

[0189] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0190] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0191] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42).

[0192] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:43, or a sequence having 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:

[0193] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0194] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0195] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0196] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 43).

[0197] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:44, or a sequence having 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:

[0198] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKF

[0199] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0200] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0201] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44).

[0202] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:45, or a sequence having 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:

[0203] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0204] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0205] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0206] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK (SEQ ID NO: 45).

[0207] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:46, or a sequence having 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:

[0208] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0209] FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA

[0210] PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0211] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK (SEQ ID NO: 46).

[0212] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO: 47, or a sequence having 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:

[0213] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVEDVSHEDPEVKF

[0214] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAP

[0215] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0216] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47).

[0217] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:48, or a sequence having 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:

[0218] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVEDVSHEDPEVKF

[0219] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAP

[0220] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0221] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 48).

[0222] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO: 49, or a sequence having 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:

[0223] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0224] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAP

[0225] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0226] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK (SEQ ID NO: 49).

[0227] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:50, or a sequence having 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:

[0228] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0229] FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPA

[0230] PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0231] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK (SEQ ID NO: 50).

[0232] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:51, or a sequence having 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:

[0233] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0234] NWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0235] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0236] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51).

[0237] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:52, or a sequence having 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:

[0238] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0239] FNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA

[0240] PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

[0241] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 52).

[0242] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:53, or a sequence having 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:

[0243] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKF

[0244] NWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0245] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0246] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53).

[0247] In some embodiments, the Fc region has an amino acid sequence comprising SEQ ID NO:54, or a sequence having 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:

[0248] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0249] NWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

[0250] IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0251] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 54).

[0252] In some preferred embodiments, the SPINK9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence as described in Table 6.

[0253] Table 6

[0254]

[0255]

[0256]

[0257] In some embodiments, the SPINK9 polypeptides described in Table 6 have 0 additional mutant SPINK9 domains, wherein a linker sequence as described herein is optionally present to connect one or more of the described domains. In certain embodiments, all domains present are connected by direct fusion.

[0258] In some embodiments, the SPINK9 polypeptides described in 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 mutant SPINK9 domains, wherein a linker sequence as described herein is optionally present to connect one or more of the described domains. In certain embodiments, all domains present are connected by direct fusion.

[0259] In some embodiments, the SPINK9 polypeptides described in Table 6 have 2 additional mutant SPINK9 domains, wherein a linker sequence as described herein is optionally present to connect one or more of the described domains. In certain embodiments, all domains present are connected by direct fusion.

[0260] In some embodiments, at least one of the mutant SPINK9 domains is linked to an additional domain (e.g., an Fc region) of a SPINK9 polypeptide via a linker sequence. In some embodiments, at least one of the mutant SPINK9 domains is linked to an additional domain (e.g., an Fc region) of a SPINK9 polypeptide via direct fusion.

[0261] In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is connected to an adjacent mutant SPINK9 domain via a linker sequence, and the mutant SPINK9 domain is connected to an additional domain (e.g., an Fc region) via a linker sequence or by direct fusion. In some embodiments, at least one mutant SPINK9 domain of a SPINK9 polypeptide is connected to an adjacent mutant SPINK9 domain via a linker sequence, and the mutant SPINK9 domain is connected to an additional domain (e.g., an Fc region) via a linker sequence or by direct fusion.

[0262] 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 shown in Table 7 below.

[0263] Table 7: Exemplary polypeptides containing mutant SPINK9 domains

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] 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 as 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).

[0275] 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 as shown in Table 7 above, wherein 1 to 3 consecutive amino acids are deleted from the N-terminus of the mutant SPINK9 domain (e.g., 1 to 3 amino acids selected from IEC).

[0276] 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 as 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).

[0277] 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.

[0278] Table 8: Exemplary polypeptides containing mutant SPINK9 domains

[0279]

[0280] The SPINK9 polypeptides may also include additional modifications that affect the properties of the SPINK9 polypeptide, such as, but not limited to, increasing desirable therapeutic properties (eg, serum half-life) and providing a simple means of purification.

[0281] In some embodiments, the SPINK9 polypeptide includes one or more bonds other than peptide bonds such that at least two adjacent amino acids are connected via bonds other than amide bonds. The use of non-peptide bonds can be used to reduce or eliminate inappropriate protein decomposition or degradation, to increase the stability of the physiological environment after administration to an individual, to increase or decrease conformational flexibility, or a combination of the foregoing. Suitable non-peptide bonds include, but are not limited to, -CH2NH-, -CH2S-, -CH2CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. Other alternatives include amide bioisosteres such as, but not limited to, 1,2,3-triazoles, oxadiazoles, imidazoles, tetrazoles, pyrazoles, indoles, pyridines, pyrazines, retro-inverted and reverse amides, ureas, olefins, fluoroolefins, trifluoroethylamine, amidines, esters, sulfonamides, phosphamides, thioamides, and carbamates (Kumari, (2020) J. Med. Chem. 63(21), 12290-12358).

[0282] In some embodiments, the SPINK9 polypeptide is modified by the addition of polyethylene glycol (PEG), PEG mimetics, polypropylene glycol, or polyoxyalkylene. Such modifications have been shown to increase physical and thermal stability, protect against enzymatic degradation, increase solubility, increase in vivo circulation half-life, reduce clearance, reduce immunogenicity and antigenicity, and reduce toxicity. Suitable PEG molecules are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2) n OR, wherein R is hydrogen or a protecting group (e.g., an alkyl or alkanol group), and wherein n is an integer from 1 to 1000. Suitable PEGs may 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 may be used n PEG can be conjugated to the SPINK9 polypeptide via a spacer comprising 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 the polyethylene glycol.

[0283] In some embodiments, the SPINK9 polypeptide is modified by changing the naturally occurring glycosylation pattern. Glycosylation can significantly affect the physical properties of proteins, and can also be important in terms of protein stability, secretion, and subcellular localization. The addition of glycosylation sites can be achieved by changing the amino acid sequence, such as replacing naturally occurring amino acids with one or more serine or threonine residues (O-linked glycosylation sites) or asparagine residues (N-linked glycosylation sites). Another way to increase the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to polypeptides. Removal of carbohydrates can be achieved chemically or enzymatically or by substitution of codons encoding glycosylated / glycosylated amino acid residues. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using a variety of endo- and exo-glycosidases.

[0284] Nucleic Acids

[0285] Provided herein are nucleic acid molecules encoding the polypeptides described herein. The nucleic acid may be present, for example, in whole cells, cell lysates, or in partially purified or substantially pure form. The nucleic acid molecules described herein can be isolated using standard molecular biology techniques and sequence information provided herein. For example, nucleic acids corresponding to nucleotide sequences encoding one or more of the polypeptides disclosed herein can be prepared by standard techniques known in the art.

[0286] The present invention provides vectors (e.g., viral vectors, e.g., adenovirus-based expression vectors) containing nucleic acid molecules described herein. Viral vectors may contain additional DNA segments that can be joined to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors or episomal mammalian vectors having a bacterial replication origin). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thereby replicated with the host genome. In addition, certain vectors can direct the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, nucleic acids that can be operably connected to one or more regulatory sequences (e.g., promoters) in the expression vector are provided herein. In some embodiments, cells transcribe the nucleic acids provided herein and thereby express antibodies, antigen-binding fragments thereof, or peptides described herein. Nucleic acid molecules may be integrated into the genome of the cell, or they may be extrachromosomal.

[0287] In some embodiments, the nucleic acid vector or recombinant adenovirus provided herein encodes a polypeptide disclosed herein or otherwise considered herein. In some embodiments, the nucleic acid vector comprises a nucleic acid sequence that is codon-optimized. In such embodiments, the coding sequence is constructed by changing the codons in each nucleic acid used to assemble the coding sequence. In general, the method for identifying a nucleotide sequence optimized for codon usage for producing a peptide comprises at least the following steps (a) to (e). In step (a), an oligomer of a portion of the coding polypeptide is provided, the portion containing a degenerate form of a codon for the amino acid encoded in the portion, wherein the oligomer is extended to provide a side-joined coding sequence with an overlapping sequence. In step (b), the oligomer is processed to achieve assembly of the coding sequence of the peptide. The recombinant peptide is included in an expression system that is operably connected to a control sequence to affect its expression. In step (c), the expression system is transfected into a culture of a compatible host cell. In step (d), the polypeptide production level of the colony obtained by the transformed host cell is tested. In step (e), at least one polypeptide is obtained from the expression system to produce the highest or satisfactory colony. The sequence of the portion of the expression system encoding the protein is determined. Further description of codon optimization is provided in U.S. Patent Publication No. US2010 / 035768, which is incorporated by reference for such teachings.

[0288] Host cells comprising nucleic acids or vectors described herein are also provided herein. The polypeptides provided herein (e.g., polypeptides comprising at least one mutant SPINK9 domain) can be separated from cells or tissue sources using standard protein purification techniques by appropriate purification procedures, can be produced by recombinant DNA technology, 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 expressing nucleotides encoding one or more peptides of the present invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for expressing heterologous peptides in recombinant hosts, chemically synthesized peptides, and in vitro translation are well known in the art and are further described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd ed., Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Vol. 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, which are incorporated herein by reference.

[0289] How to use

[0290] The present invention provides SPINK9 polypeptides and pharmaceutically acceptable salts thereof useful for treating or preventing diseases or conditions characterized by aberrant pancreatic vasodilator activity.

[0291] In certain aspects, the present invention provides a SPINK9 polypeptide or a pharmaceutically acceptable salt thereof for use as a medicament.

[0292] In certain aspects, methods for treating or preventing a disease or condition characterized by abnormal pancreatic vasodilator activity are provided. The method comprises administering an amount of a SPINK9 polypeptide or a pharmaceutically acceptable salt thereof to an individual in need, thereby treating or preventing a disease or condition characterized by abnormal pancreatic vasodilator activity. By reducing pancreatic vasodilator activity in an individual, a disease or condition characterized by abnormal pancreatic vasodilator activity is treated. In some embodiments, a therapeutically effective amount is administered to an individual.

[0293] Alternatively, in certain aspects, a SPINK9 polypeptide disclosed herein, or a pharmaceutically acceptable salt thereof, is provided for use in treating a disease or condition characterized by aberrant pancreatic vasodilator activity.

[0294] 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 treating a disease or condition characterized by aberrant pancreatic vasodilator activity.

[0295] As used herein, "a disease or condition characterized by abnormal vasodilator activity" refers to any disease or condition in which it is desirable to reduce vasodilator activity (eg, KLK5 activity). For example, it may be desirable to reduce vasodilator activity in the case of inappropriate or overactive vasodilator activity.

[0296] In certain embodiments, the disease or condition characterized by aberrant pancreatic vasodilator activity is characterized by aberrant KLK5 activity.

[0297] In certain embodiments, the disease or condition characterized by aberrant vasodilator activity is a skin disorder.

[0298] In certain embodiments, the skin disease is eczema, atopic eczema, atopic dermatitis, autosomal recessive ichthyosis, ichthyosis-like erythroderma (IE), psoriasis, UV-induced skin damage, rosacea, or a skin infection.

[0299] In certain embodiments, the disease or condition is Netherton's syndrome.

[0300] In certain embodiments, the disease or condition characterized by aberrant vasodilator activity is selected from the group consisting of: immune system hypersensitivity (atopy), hyper-IgE syndrome, allergies (including allergies to food and vacuole factors), asthma, allergic asthma, chronic inflammation, rhinitis, conjunctivitis, angioedema, eosinophilia, eosinophilic esophagitis, growth retardation, developmental failure, trichorrhexis invaginata (TI), bacterial skin infections, respiratory tract infections, systemic infections, and gastrointestinal disorders.

[0301] In certain embodiments, the disease or condition is cancer. The cancer may be selected from ovarian cancer, uterine cancer, colorectal cancer, bladder urothelial carcinoma, oral squamous cell carcinoma, breast cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, head and neck cancer, glioma, glioblastoma multiforme, and neuroblastoma.

[0302] Composition

[0303] Compositions disclosed herein include 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 present invention are suitable for treating or preventing various diseases characterized by abnormal vasodilator activity (e.g., a protease of the vasodilator-related peptidase family, specifically, abnormal KLK5 activity).

[0304] The pharmaceutical compositions provided herein may contain an amount (including a therapeutically effective amount) of SPINK9 polypeptides to treat individuals in need. 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 the polypeptide) is preferably administered in the form of a pharmaceutical composition comprising, for example, a SPINK9 polypeptide disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and unless any conventional carrier is incompatible with a substance or its derivatives, for example by producing any undesirable biological effects or otherwise interacting with any other component of the composition in a harmful manner, the use of a pharmaceutically acceptable carrier is considered to be within the scope of the present invention. In a preferred embodiment, when such pharmaceutical compositions are used for administration to humans, especially for invasive administration routes (i.e., routes that avoid transmission or diffusion through epithelial barriers, such as injection or implantation), the pharmaceutical composition is pyrogen-free, or substantially pyrogen-free. The carrier can be selected, for example, to achieve delayed release of the agent, produce a desired pH range, stabilize the Spink9 polypeptide, modulate absorption of the Spink9 polypeptide, modulate desired pharmacokinetic or pharmacodynamic properties of the Spink9 polypeptide, selectively target one or more cells, tissues or organs, or any combination of the foregoing.

[0305] Such pharmaceutically acceptable carriers include, but are not limited to, water, other liquid vehicles, solvents, diluents, dispersion media, dispersion or suspension aids, surfactants, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, antioxidants (e.g., ascorbic acid or glutathione), chelating agents, solid binders, lubricants, sugars (e.g., polydextrose, lactose, glucose and sucrose), starches (e.g., corn starch and potato starch), cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate), oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), glycols (e.g., propylene glycol), polyols (e.g., glycerol, sorbitol, mannitol and polyethylene glycol), esters (e.g., ethyl oleate and ethyl laurate), buffers (e.g., magnesium hydroxide and aluminum hydroxide, isotonic saline, Ringer's solution, phosphate buffer solution), weight molar osmotic concentration regulators and stabilizers. The choice of a pharmaceutically acceptable carrier may depend, for example, on the route of administration of the composition. The preparation of the pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be a liposome or other polymer matrix, into which the SPINK9 polypeptide disclosed herein can be incorporated, for example. Liposomes (e.g., comprising phospholipids or other lipids) are physiologically acceptable and metabolizable non-toxic carriers that can be relatively simple to prepare and administer.

[0306] The pharmaceutical composition can be provided in unit dosage form. The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of a composition sufficient to produce the desired effect, which comprises a Spink9 polypeptide alone or in combination with one or more additional therapeutic agents. It should be understood that the parameters of the unit dosage form will depend on the mode of administration, the specific Spink9 polypeptide, and the effect to be achieved. Suitable doses of Spink9 polypeptides are disclosed herein. The pharmaceutical composition can also be a solution suitable for parenteral administration, especially subcutaneous or intravenous administration, especially a liquid solution. The pharmaceutical composition can also be in lyophilized or powdered form for reconstitution with a liquid solution suitable for parenteral administration, especially subcutaneous or intravenous administration.

[0307] The pharmaceutical composition (preparation) can be administered to an individual by any of a variety of routes of administration, including, for example, parenteral administration; oral administration (e.g., in the form of a component of a liposome); administration via the oral mucosa (e.g., sublingual); transdermal administration (e.g., in the form of a patch applied to the skin); topical administration (e.g., in the form of a cream, ointment, or spray applied to the skin); or administration by inhalation. As used herein, the phrases "parenteral administration" and "parenteral administration" mean, in addition to enteral and topical administration, a mode of administration typically performed by injection or infusion, and include, but are not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, 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.

[0308] Details of administration routes and compositions suitable therefor can be found in, for example, U.S. Pat. No. 11,246,906 and International Publication No. WO 2022 / 099963.

[0309] The amount of SPINK9 polypeptide that can be combined with a pharmaceutically acceptable carrier 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 the 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.

[0310] 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 any preservatives, buffers, or propellants that may be required.

[0311] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0312] Transdermal patches have the added advantage of providing controlled delivery of the compounds of the invention to the body. Such dosage forms can be manufactured by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the amount of transdermal delivery of the compound. The rate of this flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0313] Powders and sprays may contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0314] Pharmaceutical compositions suitable for parenteral administration may comprise a combination of a Spink9 polypeptide and one or more pharmaceutically acceptable carriers, such as, but not limited to, a sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion, or a sterile powder that can be reconstituted into a sterile injectable solution or dispersion before use, which may contain antioxidants, buffers, bacteriostats, antifungal agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0315] These compositions may also contain preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial action may be ensured by including various antibacterial agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. Isotonic agents, such as sugars, sodium chloride, etc., may also need to be included in the composition. In addition, prolonged absorption of injectable pharmaceutical forms may be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0316] In some cases, to prolong the effect of the drug, it is necessary to slow down the absorption of the drug after administration (e.g., subcutaneous administration). This can be achieved by releasing the Spink9 polypeptide disclosed herein over a specified period of time using a depot injection. Depot injections are typically solid or oil-based and typically contain at least one of the pharmaceutically acceptable carriers described herein. In certain embodiments, the injectable depot form is prepared by forming a microencapsulation matrix of the target compound with a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of the drug to the polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in a liposome or microemulsion compatible with body tissues. Those of ordinary skill in the art are familiar with the possible formulations and uses of depot injections.

[0317] Administration may also be provided via implantable or biodegradable devices.

[0318] After the pharmaceutical composition has been formulated, it can be stored in a sterile container in the form of a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. These formulations can be stored in a ready-to-use form, a lyophilized form that needs to be rehydrated before use, a liquid form that needs to be diluted before use, or other acceptable forms. Preferably, the pharmaceutical composition is provided in a disposable container. Any drug delivery device can be used to deliver the pharmaceutical composition.

[0319] The present invention includes the use of pharmaceutically acceptable salts of Spink9 polypeptides in the compositions and methods of the present invention. The present invention also includes the use of buffer salts in compositions comprising Spink9 polypeptides, which are used in the methods of the compositions of the present invention. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, citrates, acetates, alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, danol, 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 therapeutic polypeptides are known in the art (Sikora et al., (2020) Pharmaceuticals, 13, 442). In certain embodiments, salts contemplated by the present invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, salts contemplated by 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 acid), carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptanoic 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, propionic 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.

[0320] 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 hydroxymethoxyphenyl (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0321] The applicable dosage of the SPINK9 polypeptide disclosed herein can be determined, at least initially, by comparing its in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art. The amount of SPINK9 polypeptide can be varied so as to obtain an amount that is effective for achieving the desired therapeutic response for a particular individual, composition, and mode of administration without being toxic to the individual.

[0322] The dosage of the 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 the treatment, other active agents administered in combination, the age, sex, weight, condition, general health and prior medical history of the individual being treated, and similar factors well known in the medical art.

[0323] A physician generally skilled in the art can determine and prescribe the amount of the pharmaceutical composition required. For example, the physician may start administration of the pharmaceutical composition or compound at a level below that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A larger total dose may be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th Edition, 1814-1882, incorporated herein by reference).

[0324] In certain embodiments, pharmaceutical compositions for administration, particularly subcutaneous and intravenous administration, comprise the following dosages of Spink9 polypeptide: about 0.1 mg to about 50 mg; about 0.1 mg to about 40 mg; about 0.1 mg to about 30 mg; about 0.1 mg to about 20 mg; about 0.1 mg to about 15 mg; about 0.1 mg to about 10 mg; about 0.1 mg to about 8 mg; about 0.1 mg to about 6 mg; about 0.1 mg to about 4 mg; about 0.1 mg to about 2 mg; about 0.1 mg to about 1 mg; about 0.5 mg to about 50 mg; about 0.5 mg to about 40 mg; about 0.5 mg to about 30 mg; about 0.5 mg to about 20 mg; about 0.5 mg to about 15 mg; about 0.5 mg to about 10 mg; about 0.5 mg to about 8 mg; about 0.5 mg to about 6 mg; about 0.5 mg to about 4 mg; about 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.

[0325] The dose of the Spink9 polypeptide to be administered, in particular for subcutaneous and intravenous administration, can also be expressed in μg / kg. Exemplary dosages of Spink9 polypeptides are in the following ranges in μg / kg: 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; about 1 μg / kg to about 10 μg / kg; about 1 μg / kg to about 5 μg / kg; about 5 μg / kg to about 500 μg / kg; about 5 μg / kg to about 250 μg / kg; about 5 μg / kg to about 100 μg / kg; about 5 μg / kg to about 50 μg / kg; about 5 μg / kg to about 25 μg / kg; about 5 μg / kg to about 15 μg / kg; about 5 μg / kg to about 10 μg / kg; about 15 μg / kg to about 500 μg / kg; about 15 μg / kg to about 250 μg / kg; about 15 μg / kg to about 100 μg / kg; about 15 μg / kg to about 50 μg / kg; about 15 μg / kg to about 25 μg / kg; about 50 μg / kg to about 500 μg / kg; about 50 μg / kg to about 250 μg / kg; about 50 μg / kg to about 100 μg / kg; 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; about 400 μg / kg to about 500 μg / kg. Furthermore, the dosage in μg / kg can be calculated by dividing the fixed dose as described herein by 60 to 70 kg for adult subjects or 15 to 50 kg for pediatric subjects.

[0326] In certain embodiments, the Spink9 polypeptide, alone or as part of a pharmaceutical composition, is administered according to a course of treatment. A course of treatment may extend for 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, a course of treatment is for the remainder of an individual's lifespan.

[0327] An individual may receive a single treatment course or multiple (i.e., 1, 2, 3, 4, 5 or more) treatment courses. When an individual receives multiple treatment courses, such multiple treatment courses may be continuous (e.g., starting a second treatment course immediately after the completion of a first treatment course), or such multiple treatment courses may be non-continuous and separated by non-treatment intervals (e.g., starting a third treatment course 1 month after the completion of a second treatment course). A combination of continuous and non-continuous treatment courses may also be used (e.g., starting a third treatment course 1 month after the completion of a second treatment course, and starting a second treatment course immediately after the completion of a first treatment course). In certain aspects, 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 longer.

[0328] Dosing regimens can be used with treatment courses. When multiple treatment courses are administered to an individual, the dosing regimen for each treatment course can be the same, or the dosing regimen for one or more treatment courses can be different. In one embodiment, the dosing regimen comprises administering a Spink9 polypeptide during the following treatment course: 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 day, 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.

[0329] Preferably, the individual receives multiple treatment courses. In certain embodiments, the Spink9 polypeptide is administered by a parenteral administration route. In certain embodiments, the Spink9 polypeptide is administered by subcutaneous administration. In certain embodiments, the Spink9 polypeptide is administered by intravenous administration.

[0330] When an individual receives a course of treatment by subcutaneous administration, the Spink9 polypeptide is injected at the same site on the individual for each dosing regimen (e.g., upper arm) or at different sites on the individual for one or more dosing regimens (e.g., upper arm and abdomen). In certain embodiments, subcutaneous administration is accomplished via the use of an implantable device.

[0331] In certain embodiments, the SPINK9 polypeptides disclosed herein may be administered alone or in combination with another type of therapeutic agent, such as other agents suitable for treating or preventing diseases or conditions characterized by abnormal pancreatic vasodilator activity. The SPINK9 polypeptides disclosed herein may also be administered in combination with other therapeutic agents, such as other agents suitable for treating or preventing diseases or conditions characterized by abnormal KLK5 activity. In certain embodiments, the SPINK9 polypeptides disclosed herein may also be administered in combination with one or more inhibitors of KLK family members (specifically, KLK family members downstream of KLK5 (such as, but not limited to, KLK7 and KLK14)). In certain embodiments, the SPINK9 polypeptides disclosed herein may also be administered in combination with one or more other therapeutic agents suitable for treating or preventing Netherton syndrome. For example, and without being bound by theory or method, "combined administration" may refer to any form of administration of the SPINK9 polypeptide disclosed herein with other therapeutic agents, 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 vivo (e.g., the SPINK9 polypeptide and the other therapeutic agent are effective simultaneously in the patient, which may include a synergistic effect). The SPINK9 polypeptide and other different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, individuals receiving such treatment may benefit from the combined effects of different therapeutic agents.

[0332] Examples

[0333] Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0334] Example 1: Construction and expression of SPINK9 polypeptide vector

[0335] The SPINK9 polypeptide was produced recombinantly as follows. First, a cDNA sequence encoding the desired SPINK9 domain (e.g., amino acids 20 to 86 of SEQ ID NO: 1) fused to the C-terminal IgG Fc fragment was designed. The codon usage of the mammalian expression system was optimized using the GeneArt codon optimizer, and the Kozak sequence suggested by it was used. The cDNA was synthesized and the nucleotide sequence was confirmed. The cDNA was cloned into the pcDNA3.4 expression vector using EcoR1 and HindIII sites. The pcDNA3.4 vector uses the full-length human cytomegalovirus (CMV) immediate early promoter for achieving high gene expression levels, and a woodchuck post-transcriptional regulatory element (WPRE) downstream of the cloning site to enhance transcript expression. The mouse IgG heavy chain signal peptide (MGWSCIILFLVATATGVHS) was added to the N-terminus of the SPINK9 polypeptide. ) Or wild-type SPINK9 signal peptide (such as SEQ ID NO:1 amino acid 1-19) to ensure secretion into the cell culture medium. During translocation, the signal peptide is cleaved. Expi293 cells (A14527, ThermoFisher) or CHO-S cells (R80007, ThermoFisher) are used to express SPINK9 polypeptides. For expression, Expi293 cells are transiently transfected with expression vectors and cell cultures are collected. SPINK9 polypeptides are purified from cell culture medium using HiTrap protein GHP 5ml columns (17-0405-01, GE Healthcare). The filtered cell culture medium is loaded on the protein G column according to the protein G protocol, and PBS balance columns (binding buffer) and 0.7% acetic acid (elution buffer) are used to elute the SPINK9 polypeptides. The eluate is monitored by measuring the absorbance at 280nm. After purification, the SPINK9 polypeptide is stored in PBS, and therefore directly after elution, the buffer is replaced by dialysis or by a desalting column with an appropriate molecular weight cutoff. The 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. The protein concentration was measured using a micro-spectrophotometer at an A280 setting. The expression of the desired SPINK9 polypeptide was further confirmed under reducing and non-reducing conditions by SDS page and Western blot assays.

[0336] Example 2: The inhibitory activity of SPINK9 peptides on KLK5 was evaluated.

[0337] The effect of SPINK9 polypeptide on KLK5 enzyme activity was evaluated. The assay was performed at 22°C by incubating 20 nM KLK5 (pH 7.5 and pH 5.5) in 100 mM NaH2PO4, 1 mM CHAPS with SPINK9 polypeptide (half-log dilution series from 0.3 nM to 300 nM) or incubating the assay buffer as a control for 10 minutes. The reaction was then initiated by adding 100 μM of the substrate BOC-VPR-AMC (R&D Systems, Minneapolis Minnesota) in a total reaction volume of 100 μL. After incubation for 15 minutes, the culture plate was excited at 340 nm and read at 460 nM by a Synergy Neo microplate reader ( Fluorescence emission was measured in 1% 4 ... 50 The results are shown in Table 9. Wild-type Spink9 (SEQ ID NO: 2) exhibited IC values ​​of 80 nM and 685 nM when binding to KLK5 at pH 7.5 and 5.5, respectively. 50 .

[0338] Table 9 - Inhibition of KLK5 enzyme activity by SPINK9 peptide

[0339]

[0340]

[0341] *n=1; ND=not determined

[0342] Example 3: The inhibitory activity of SPINK9 peptides on KLK7 and KLK14 was evaluated.

[0343] The effect of SPINK9 polypeptide on KLK7 and KLK14 enzyme activity was evaluated. Both rhKLK7 and rhKLK14 were obtained in zymogen form and each required activation by thermolysin. RhKLK7 or rhKLK14 was diluted to 200 mg / mL in activation buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 7.5) and mixed with the same volume of 20 mg / mL thermolysin in the same buffer. Activation was performed at 37°C for 1 hour (rhKLK14) or 2 hours (rhKLK7), and the reaction was stopped by adding 50 mM EDTA.

[0344] The KLK14 assay was performed as described above in Example 2, except that the concentration of active KLK14 was 0.2 mg / ml. Enzyme activity was measured by fluorescence emission and the IC was calculated as described above in Example 2. 50 .

[0345] 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 of SPINK9 peptide (half-log dilution series from 0.3 nM to 300 nM) or assay buffer as a control for 15 minutes at 22°C. The reaction was initiated by adding 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 incubation for 15 minutes, the plates were excited at 320 nm and read at 405 nM using a Synergy Neo microplate reader ( Fluorescence emission was measured at 4 °C in Winooski, Vermont. IC was calculated as described in Example 2 above. 50 .

[0346] The results for both KLK7 and KLK14 are shown in Table 10.

[0347] Table 10 - Inhibitory effect of SPINK9 polypeptide on KLK7 and KLK14 enzyme activities

[0348]

[0349] *Results are presented as IC at 316nM SPINK9 peptide 50 or inhibition percentage

[0350] Example 4: Evaluation of human serum stability.

[0351] The exemplary SPINK9 polypeptide SPINK9-112.2K62G.Fc (SEQ ID NO: 76) was diluted in freshly collected human serum (1:9) and distributed into 200 μL aliquots. Five aliquots of the sample were then kept in an incubator set to 37°C, 5% CO2. Samples were collected and snap-frozen on days 0, 1, 4, 7, and 14, and then kept at -80°C until assayed. The binding activity of the tested samples to recombinant human KLK5 C-6His (Novoprotein, catalog number C415) was assessed by ELISA as described below.

[0352] Binding of SPINK9-112.2K62G.Fc after serum incubation was determined by ELISA. Plates were pre-coated with 100 μl / well of THE in a refrigerator set at 4°C. TM His-tagged mAb (2 μg / ml) (Genscript; Catalog No.: A00186) overnight. After blocking with 200 μl / well of 0.5% casein for 1 hour, the culture plates were washed 3 times with 1×PBST. Next, 0.25 μg / ml recombinant human KLK5 C-6His was added to the culture plates at a volume of 100 μl / well and incubated at ambient temperature for 1 hour. After washing the culture plates 3 times with 1×PBST, various concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.0013, 0.00026, 0.000051 and 0.000010 nM) of SPINK9-112.2K62G.Fc and dilution buffer (0.25% casein) were added to the culture plates at a volume of 100 μl / well and incubated for 2 hours at ambient temperature. After washing the plate 3 times with 1×PBST, 100 μL / well of HRP-labeled goat anti-human IgG antibody (1:5000) (Bethyl; Catalog No.: A80-304P-16) was added to each well with dilution buffer (0.25% casein) and incubated at ambient temperature for 1 hour. After washing 6 times with 1×PBST, color was developed for 4 minutes by dispensing 100 μL / well of TMB substrate (Life Technologies; Catalog No.: 00203), and the reaction was terminated by adding 100 μL / well 2M HCl. The absorbance at 450 and 540 nm was read using a microplate reader (SpectraMax M5e). EC was calculated by four-parameter nonlinear regression determination using GraphPadPrism software. 50 value.

[0353] The binding ability of SPINK9-112.2K62G.Fc incubated with human serum at different time points to recombinant human KLK5 C-6His was tested. The results of the serum stability test (Table 11) showed that SPINK9-112.2K62G.Fc incubated with serum maintained binding activity to KLK5 for up to two weeks.

[0354] Table 11 - Human serum stability test results of SPINK9-112.2K62G.Fc

[0355]

[0356] Table 12 shows human serum stability and affinity testing of other Spink9 polypeptides performed according to the procedures described above.

[0357] Table 12 - Human serum stability test results of Spink9 polypeptide

[0358]

[0359] Example 5: Binding to human KLK5 at 7.4 by surface plasmon resonance (SPR).

[0360] SPR assays were performed using Biacore T200 (GE Healthcare) with S-series CM5 sensor chips (GE Healthcare).Anti-human IgG Fc monoclonal antibody was diluted to 25 μg / ml in immobilization buffer (10 mM sodium acetate, pH 5.0).

[0361] The CM5 sensor chip was activated by an activator mixture (prepared by mixing 400 mM EDC and 100 mM NHS just before injection) at a flow rate of 10 μL / min for 420 s. Subsequently, 25 μg / mL of anti-human IgG Fc monoclonal antibody in fixed buffer was injected into the Fc2 sample channel at a flow rate of 10 μL / min for 420 s, resulting in a fixed level of approximately 7000 RU. The sensor chip was deactivated by 1 M ethanolamine hydrochloride-NaOH (GE Healthcare) at a flow rate of 10 μL / min for 420 s. The reference surface channel Fc1 was prepared in the same manner.

[0362] Spink9 polypeptide was diluted to m0.1 to 0.7 μg / mL in running buffer (1×HBS-EP; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) and then injected into channel Fc2 at a flow rate of 10 μL / min to achieve capture levels of approximately 35.6 to 64.5 RU.

[0363] KLK5 protein (Novoprotein; catalog number C415) was diluted to 5 to 6 concentrations as described below with operating buffer (1×HEPES; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4). KLK5 protein was injected into the Fc1 and Fc2 channels at a flow rate of 30 μL / min, with an association phase of 120 s followed by a dissociation of 300 s (both the association and dissociation phases were performed in operating buffer). Increasing concentrations of KLK5 assays were measured. After each cycle of the interaction assay, the sensor chip surface was regenerated for 30 s with 3M MgCl as injection buffer at a flow rate of 10 μL / min to remove ligand and any non-bound KLK5.

[0364] Spink9 WT - 100, 50, 25, 12,5 and 6.25 nM KLK5

[0365] Spink9-30.Fc (SEQ ID NO: 55) - 65, 32.5, 16.25, 8.125, 4.063 and 2.032 nM KLK5

[0366] Spink9-112.K62G.Fc (SEQ ID NO:56) - 50, 25, 12, 5, 6.25 and 3.125 nM KLK5

[0367] Spink9-183.Fc (SEQ ID NO:57) - 100, 50, 25, 12, 5, 6.25 and 3.125 nM KLK5

[0368] Spink9-186.Fc (SEQ ID NO:58) - 100, 50, 25, 12, 5, 6.25 and 3.125 nM KLK5

[0369] Spink9-189.Fc (SEQ ID NO:59) - 100, 50, 25, 12, 5, 6.25 and 3.125 nM KLK5

[0370] Spink9-236.Fc (SEQ ID NO:60) - 100, 50, 25, 12, 5, 6.25 and 3.125 nM KLK5

[0371] The sensorgrams for the reference channel and the buffer channel 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 ​​of the tested polypeptides. D value.

[0372] Table 13 - Binding affinity of Spink9 peptide to human KLK5

[0373] <![CDATA[K D (M)]]> Spink9WT 9.90E-08 Spink9-30 (SEQ ID NO:55) 9.09E-11 Spink9-112.K62G (SEQ ID NO: 56) 7.85E-14 Spink9-183 (SEQ ID NO:57) 2.16E-11 Spink9-186 (SEQ ID NO:58) 1.89E-11 Spink9-189 (SEQ ID NO:59) 1.52E-10 Spink9-236 (SEQ ID NO:60) 2.909E-11

[0374] Example 6: Binding to human FcRn at pH 6.0 and 7.4 by surface plasmon resonance (SPR).

[0375] Biacore 8K was used together with S-series CM5 sensor chips (Cytiva) to perform SPR assays. The CM5 sensor chips were activated by an activator mixture (prepared by mixing 400 mM EDC and 100 mM NHS just before injection) at a flow rate of 10 μL / min for 420 s. 10 μg / mL 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 then injected into Fc2 at a flow rate of 10 μl / min for 60 s. The reference channel Fc1 was blocked. The sensor chip was deactivated by 1 M ethanolamine-HCl (Cytiva) at a flow rate of 10 μL / min for 420 s.

[0376] 1×PBST (50 mM Na2HPO4 / NaH2PO4, 150 mM NaCl, 0.05% Tween-20, pH 6.0 or pH 7.4) (AcrobioSystems; Catalog No.: FCM-H5286) containing 8 concentrations (46.875, 93.75, 187.5, 375, 750, 1500, 3000 and 6000 nM) of the assay human FcRn and operating buffer (1×PBST, pH 6.0) were injected into Fc1 and Fc2 at a flow rate of 30 μL / min, with an association phase of 60 s, followed by a dissociation phase of 90 s. After each dissociation phase, 1×PBS (pH 7.4) as a regeneration buffer was injected into the flow cell at a flow rate of 10 μL / min for 45 s.

[0377] The sensorgrams for the reference channel and the buffer channel 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 ​​of the tested polypeptides. D value.

[0378] Table 14 - Binding affinity of mutant Spink9 polypeptides to human FcRn

[0379]

[0380] Example 7: Single-dose PK study of SPINK9 peptide in cynomolgus monkeys.

[0381] Eight cynomolgus macaques (4 males and 4 females) weighing between 2.5 and 3.5 kg were used in this study. The animals were supplied with approximately 120 grams of certified monkey food (Beijing Vital Keao Feed Co., Ltd. Beijing, PR China) daily. The monkeys received fresh fruit daily. The animals were provided with reverse osmosis purified water ad libitum by an automated water supply system.

[0382] Four (4) male and four (4) female cynomolgus macaques were randomly assigned to four (4) groups (one male and one female cynomolgus macaque 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 via intravenous (IV) infusion over 45 minutes at a single dose of 10 mg / kg 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). Spink9-183 (SEQ ID NO: 57) was administered to animals in Group 4. Animals were randomly assigned to each group based on body weight. Animals were weighed prior to dosing and the amount of the dose was determined based on the body weight recorded on the dose record sheet. The start date of the first dosing was recorded as Day 0.

[0383] In all groups, animals had at least 200 μL of blood samples collected from the head or saphenous vein at the sampling time points. For samples collected within the first hour of dosing, ±1 minute was acceptable. For the remaining time points, samples were taken within 5% of the scheduled time. All blood samples were collected into commercially available tubes containing a coagulant. Subsequently, the tubes containing the blood samples were maintained at room temperature for 30 minutes and then centrifuged. The samples were centrifuged and serum was collected for PK assays. Samples were collected at the following time points: before dosing (Day -1) and at 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) after dosing.

[0384] Observe the body weight and feeding status of the animals (twice a day). Cage-side observations of general health and appearance were performed once a day. Animals were observed before and after dosing and at each sample collection time point. Body weight and feeding status were recorded. Each animal was weighed twice during the environmental adaptation period and once before the test (day 0). After the test article was applied, each animal was weighed twice a week from day 0 to day 28. Daily food consumption of all animals was also measured from day 0 to day 28.

[0385] PK method

[0386] The concentration of the test article in serum was determined using 2 different assay bioassay ELISA methods, the Fc+Fc assay and the KLK5+Fc assay.

[0387] Fc+Fc: Briefly, 96-well ELISA plates were incubated overnight at 4°C with 1.0 μg / mL goat anti-human IgG (Southern Biotech; Catalog No.: 2049-01) solution. After washing and blocking, serially diluted plasma samples were added, followed by the addition of biotinylated 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) substrates were used for color development. The reaction was stopped after about 5 to 10 minutes by adding 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer (SpectraMax M5e). Using the standard curve, the OD values ​​of the samples were used to derive the plasma concentration of the test article. The detection limit of this ELISA method was 0.391 ng / mL.

[0388] KLK5+Fc: Briefly, 96-well ELISA plates were incubated overnight at 4°C with 1.0 μg / mL His tag antibody (Sino Biological; Catalog No.: A00186-100). The plates were washed and 0.25 μg / mL recombinant human KLK5 C-6His (Novoprotein; Catalog No.: C415) was added to each well. After washing and blocking, serially diluted plasma samples were added, followed by biotinylated goat anti-human IgG (dilution factor: 8000) (Southern Biotech; Catalog No.: 2049-08). Streptavidin-HRP (dilution factor: 20000) (Thermo-Fischer; Catalog No.: 21127) and TMB (Life Technologies; Catalog No.: 002023) substrates were used for color development. The reaction was stopped after about 5 to 10 minutes by adding 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer (SpectraMax M5e). The OD values ​​of the samples were used to derive the plasma concentration of the test article using a standard curve. The detection limit of this ELISA method was 0.195 ng / mL.

[0389] Non-compartmental pharmacokinetic determinations of serum concentrations of the four test articles were performed using Phoenix Win Nonlin software (version 8.1, Pharsight, Mountain View, CA). PK parameters were derived using the linear / log trapezoidal rule.

[0390] The pharmacokinetic parameters of the test articles for the Fc+Fc method and the KLK5+Fc method are provided in Tables 15 and 16, respectively, and are shown in Tables 15 and 16, respectively. Figure 2 and 3 middle.

[0391] Table 15 - Summary of pharmacokinetic parameters: Fc+Fc

[0392]

[0393] Table 16 - Summary of Pharmacokinetic Parameters: KLK5+Fc

[0394]

[0395]

[0396] All animals expressed normal results. No formulation-related effects were found on body weight or weight gain ( Figure 1 ). No formulation-related effects on food consumption were found during the study period.

[0397] Pharmacokinetic results confirmed that the Spink9-112.2K62G.Fc peptide showed the most favorable results, with an average half-life of 128 hours in the Fc+Fc assay and an average AUC 0-最后 The average Cl _obs The mean half-life in the KLK5+Fc assay was 131 hours, and the mean AUC 0-最后 The average Cl _obs It is 1.03mL / h / kg.

[0398] Equivalent

[0399] Although specific embodiments of the present invention have been discussed, the above description is illustrative and not restrictive. Various variations of the present invention will become apparent to those skilled in the art upon reviewing this specification and the following claims. The full scope of the present invention should be determined by reference to the full scope of the claims and their equivalents, and the specification and such variations.

[0400] References incorporated

[0401] All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, waivers or disclaimers of subject matter, and to the extent the incorporated material is inconsistent with the language disclosed herein, in which case the language of the present disclosure controls.

Claims

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

2.

2. The polypeptide of claim 1, wherein the polypeptide binds to and inhibits active pancreatic vasodilator-related peptidase.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide inhibits the activity of vasodilator-related peptidase 5 (KLK5).

4. A polypeptide according to any one of claims 1 to 3, wherein the second amino acid sequence comprises at least one amino acid substitution relative to position 22, 24, 25, 26, 28, 29, 30 or any combination thereof of SEQ ID NO: 2, and optionally comprises one or more amino acid changes relative to position 21, 23, 27, 31, 32, 33 and 34 of SEQ ID NO:

2.

5. according to the polypeptide described in any one of claims 1 to 3, wherein the first amino acid sequence optionally comprises the amino acid at position 1, 2 and 3 of SEQ ID NO:2, and the second amino acid sequence comprises at least one amino acid substitution relative to position 22, 24, 25, 26, 28, 29, 30 or any combination thereof of SEQ ID NO:2, and optionally comprises one or more amino acid changes relative to position 21, 31, 32, 33 and 34 of SEQ ID NO:

2.

6. A polypeptide according to any one of claims 1 to 3, wherein the first amino acid sequence comprises a deletion of the amino acid at positions 1, 2 and 3 of SEQ ID NO:2, and the second amino acid sequence comprises at least one amino acid substitution relative to positions 22, 24, 25, 26, 28, 29, 30 or any combination thereof of SEQ ID NO:2, and optionally comprises one or more amino acid changes relative to positions 21, 31, 32, 33 and 34 of SEQ ID NO:

2.

7. A polypeptide according to any one of claims 1 to 6, wherein the second amino acid sequence comprises a substitution of Glu, Met, Gln, Ala, Leu, Val, Asp, Asn, Ser, Lys or His amino acid relative to 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.

8. The polypeptide of claim 7, wherein the second amino acid sequence comprises a substitution of Glu, Thr, Val, Leu, Ala, Ile, Phe, Ser or Tyr amino acid relative to 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.

9. The polypeptide of claim 7 or 8, wherein the second amino acid sequence comprises a substitution of Leu, Ile, His, Glu, Trp, Ser, Ala, Val, Tyr, Asp or Met amino acid relative to the amino acid at position 25 of SEQ ID NO: 2, or the amino acid at position 25 of SEQ ID NO: 2 is Arg.

10. The polypeptide according to any one of claims 7 to 9, wherein the second amino acid sequence comprises a substitution of Tyr, Trp, Met, His, Lys or Asn amino acid for the amino acid at position 26 relative to SEQ ID NO: 2, or the amino acid at position 26 of SEQ ID NO: 2 is Phe.

11. The polypeptide according to any one of claims 7 to 10, wherein the second amino acid sequence comprises a substitution of Thr, Ser or Gly amino acid for the amino acid at position 28 relative to SEQ ID NO: 2, or the amino acid at position 28 of SEQ ID NO: 2 is His.

12. The polypeptide according to any one of claims 7 to 11, wherein the second amino acid sequence comprises a substitution of an Arg or Lys amino acid for the amino acid at position 29 relative to SEQ ID NO: 2, or the amino acid at position 29 of SEQ ID NO: 2 is His.

13. The polypeptide according to any one of claims 7 to 12, wherein the second amino acid sequence comprises a substitution of a Glu or Asp amino acid for the amino acid at position 30 relative to SEQ ID NO: 2, or the amino acid at position 30 of SEQ ID NO: 2 is Met.

14. The polypeptide according to any one of claims 1 to 6, wherein the second amino acid sequence comprises a substitution of a Glu or Met amino acid for the amino acid at position 22 relative to SEQ ID NO:

2.

15. The polypeptide of claim 14, wherein the second amino acid sequence comprises a substitution of a Glu or Thr amino acid for the amino acid at position 24 relative to SEQ ID NO:

2.

16. The polypeptide according to claim 14 or 15, wherein the second amino acid sequence comprises a substitution of a Leu, Ile, His, Glu or Trp amino acid for the amino acid at position 25 relative to SEQ ID NO:

2.

17. The polypeptide according to any one of claims 14 to 16, wherein the second amino acid sequence comprises a substitution of a Tyr or Trp amino acid for the amino acid at position 26 relative to SEQ ID NO:

2.

18. The polypeptide according to any one of claims 14 to 17, wherein the second amino acid sequence comprises a substitution of Thr, Ser or Gly amino acid for the amino acid at position 28 relative to SEQ ID NO:

2.

19. The polypeptide according to any one of claims 14 to 18, wherein the second amino acid sequence comprises a substitution of an Arg or Lys amino acid for the amino acid at position 29 relative to SEQ ID NO:

2.

20. The polypeptide according to any one of claims 14 to 19, wherein the second amino acid sequence comprises a substitution of a Glu or Asp amino acid for the amino acid at position 30 relative to SEQ ID NO:

2.

21. The polypeptide according to any one of claims 7 to 20, wherein the third amino acid sequence comprises a substitution of a Gly amino acid for the amino acid at position 43 relative to SEQ ID NO:

2.

22. The polypeptide of any one of claims 7 to 21, wherein the second amino acid sequence comprises a substitution of Thr-Arg-Glu; Thr-Arg-Asp; Thr-Lys-Glu; Thr-Lys-Asp; Ser-Arg-Glu; or Gly-Arg-Asp amino acid for amino acids at positions 28-30 relative to SEQ ID NO:

2.

23. The polypeptide of any one of claims 1 to 3, wherein the at least one mutant SPINK9 domain comprises an amino acid sequence selected from SEQ ID NO: 4-23 or 115-134.

24. The polypeptide of any one of claims 1 to 22, wherein the polypeptide comprises two to twenty mutant SPINK9 domains.

25. The polypeptide of claim 24, wherein each of the two to twenty mutant SPINK9 domains comprises an independently selected amino acid sequence of any one of SEQ ID NOs: 4-23 or 115-134.

26. The polypeptide of any one of claims 1 to 22, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain and a second mutant SPINK9 domain; and each of the first mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of any one of SEQ ID NOs: 4-23 or 115-134.

27. The polypeptide of claim 26, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

28. The polypeptide of claim 26, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

29. The polypeptide of any one of claims 1 to 22, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain, a second mutant SPINK9 domain, and a third mutant SPINK9 domain; and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, and the third mutant SPINK9 domain comprises an independently selected amino acid sequence of any one of SEQ ID NOs: 4-23 or 115-134.

30. The polypeptide of claim 29, wherein each of the mutant SPINK9 domains are identical.

31. The polypeptide of claim 29, wherein each of the mutant SPINK9 domains is different.

32. A polypeptide according to any one of claims 1 to 22, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, wherein the first polypeptide chain comprises a first mutant SPINK9 domain and a second mutant SPINK9 domain and the second polypeptide chain comprises a third mutant SPINK9 domain and a fourth mutant SPINK9 domain, and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, the third mutant SPINK9 domain and the fourth mutant SPINK9 domain comprises an independently selected amino acid sequence of SEQ ID NO: 4-23 or 115-134.

33. The polypeptide of claim 32, wherein each of the mutant SPINK9 domains are identical.

34. The polypeptide of claim 32, wherein each of the mutant SPINK9 domains is different.

35. The polypeptide of any one of claims 24 to 34, wherein adjacent mutant SPINK9 domains are connected by direct fusion, a linker sequence, or any combination thereof.

36. The polypeptide of claim 35, wherein at least one mutant SPINK9 domain is linked to an adjacent mutant SPINK9 domain via a linker sequence.

37. The polypeptide of claim 35, wherein at least one mutant SPINK9 domain is linked to an adjacent mutant SPINK9 domain by direct fusion.

38. A polypeptide according to any one of claims 35 to 37, wherein the linker sequence comprises a cleavage site.

39. The polypeptide of claim 38, wherein the cleavage site is recognized by a subtilisin-like proprotein convertase.

40. The polypeptide of any one of claims 1 to 22, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, 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 mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of SEQ ID NO: 4-23 or 115-134.

41. The polypeptide of claim 40, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

42. The polypeptide of claim 40, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

43. A polypeptide comprising at least one mutant SPINK9 domain, wherein 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 LysLeu Pro Pro X1Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro Ile Cys Gly Ser Asp Gly LysThr Tyr Cys(SEQ ID NO:3); The prerequisite is that the amino acid sequence represented by Formula I is not SEQ ID NO:

2.

44. The polypeptide of claim 43, wherein the polypeptide binds to and inhibits active vasodilator-related peptidase.

45. The polypeptide of claim 43 or 44, wherein the polypeptide inhibits the activity of vasodilator-related peptidase 5 (KLK5).

46. ​​The polypeptide of claim 45, wherein the inhibition is selective for KLK5 or specific for KLK5.

47. A polypeptide according to any one of claims 43 to 46, wherein 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.

48. The polypeptide of any one of claims 43 to 47, wherein 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.

49. The polypeptide of any one of claims 43 to 48, wherein 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.

50. The polypeptide of any one of claims 43 to 49, wherein 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.

51. The polypeptide of any one of claims 43 to 50, wherein X5 is selected from: i) His, Thr, Ser or Gly; ii) His or Thr; iii) Thr, Ser or Gly; or iv) Thr.

52. The polypeptide of any one of claims 43 to 51, wherein X6 is selected from: i) His, Arg or Lys; ii) His or Arg; iii) Arg or Lys; or iv) Arg.

53. The polypeptide of any one of claims 43 to 52, wherein X7 is selected from: i) Met, Glu or Asp; ii) Met or Glu; iii) Glu or Asp; or iv) Glu.

54. The polypeptide of any one of claims 43 to 53, wherein X8 is selected from: i) Lys or Gly; ii) Lys; or iii) Gly.

55. The polypeptide of any one of claims 43 to 54, wherein X5-X6-X7 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.

56. The polypeptide of any one of claims 43 to 55, wherein the polypeptide comprises two to twenty independently selected mutant SPINK9 domains; and each mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula I or SEQ ID NO: 4-23.

57. The polypeptide of any one of claims 43 to 55, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain and a second mutant SPINK9 domain; and each of the first mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula I or SEQ ID NO: 4-23.

58. The polypeptide of claim 57, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

59. The polypeptide of claim 57, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

60. The polypeptide of any one of claims 43 to 55, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain, a second mutant SPINK9 domain, and a third mutant SPINK9 domain; and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, and the third mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula I or SEQ ID NO: 4-23.

61. The polypeptide of claim 60, wherein each of the mutant SPINK9 domains are identical.

62. The polypeptide of claim 60, wherein each of the mutant SPINK9 domains is different.

63. A polypeptide according to any one of claims 43 to 55, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, wherein the first polypeptide chain comprises a first mutant SPINK9 domain and a second mutant SPINK9 domain and the second polypeptide chain comprises a third mutant SPINK9 domain and a fourth mutant SPINK9 domain, and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, the third mutant SPINK9 domain and the fourth mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula I or SEQ ID NO: 4-23.

64. The polypeptide of claim 63, wherein each of the mutant SPINK9 domains are identical.

65. The polypeptide of claim 64, wherein each of the mutant SPINK9 domains is different.

66. The polypeptide of any one of claims 56 to 65, wherein adjacent mutant SPINK9 domains are connected by direct fusion, a linker sequence, or any combination thereof.

67. The polypeptide of claim 66, wherein at least one mutant SPINK9 domain is linked to an adjacent mutant SPINK9 domain via a linker sequence.

68. The polypeptide of claim 66, wherein at least one mutant SPINK9 domain is linked to an adjacent mutant SPINK9 domain by direct fusion.

69. A polypeptide according to any one of claims 66 to 68, wherein the linker sequence comprises a cleavage site.

70. The polypeptide of claim 69, wherein the cleavage site is recognized by a subtilisin-like proprotein convertase.

71. The polypeptide of any one of claims 43 to 55, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, wherein the first polypeptide chain contains a first mutant SPINK9 domain and the second polypeptide chain contains a second mutant SPINK9 domain, and each of the first mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula I or SEQ ID NO: 4-23.

72. The polypeptide of claim 71, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

73. The polypeptide of claim 71, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

74. A polypeptide comprising at least one mutant SPINK9 domain, wherein the at least one mutant SPINK9 domain comprises an amino acid sequence represented by Formula IV; X9 X 10 X 11 Ala Lys Gln Thr Lys Gln Met Val Asp CysSer His Tyr Lys Lys Leu Pro Pro X1 Gln X2 X3 X4 Cys X5 X6 X7 Tyr Asp Pro IleCys Gly Ser Asp Gly Lys Thr Tyr NO:136); The prerequisite is that the amino acid sequence represented by Formula IV is not SEQ ID NO:

2.

75. The polypeptide of claim 74, wherein the polypeptide binds to and inhibits active vasodilator-related peptidase.

76. The polypeptide of claim 74 or 75, wherein the polypeptide inhibits the activity of vasodilator-related peptidase 5 (KLK5).

77. The polypeptide of claim 76, wherein the inhibition is selective for KLK5 or specific for KLK5.

78. The polypeptide of any one of claims 74 to 67, wherein 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.

79. The polypeptide of any one of claims 74 to 78, wherein 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.

80. The polypeptide of any one of claims 74 to 79, wherein 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.

81. The polypeptide of any one of claims 74 to 80, wherein 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.

82. The polypeptide of any one of claims 74 to 81, wherein X5 is selected from: i) His, Thr, Ser or Gly; ii) His or Thr; iii) Thr, Ser or Gly; or iv) Thr.

83. The polypeptide of any one of claims 74 to 82, wherein X6 is selected from: i) His, Arg or Lys; ii) His or Arg; iii) Arg or Lys; or iv) Arg.

84. The polypeptide of any one of claims 74 to 83, wherein X7 is selected from: i) Met, Glu or Asp; ii) Met or Glu; iii) Glu or Asp; or iv) Glu.

85. The polypeptide of any one of claims 74 to 84, wherein X8 is selected from: i) Lys or Gly; ii) Lys; or iii) Gly.

86. The polypeptide of any one of claims 74 to 85, wherein X9 is selected from: i) absent; or ii) Ile.

87. A polypeptide according to any one of claims 74 to 86, wherein X 10 Selected from: i) absent; or ii) Glu.

88. A polypeptide according to any one of claims 74 to 87, wherein X 11 Selected from: i) absent; or ii) Cys.

89. The polypeptide of any one of claims 74 to 88, wherein X5-X6-X7 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.

90. The polypeptide of any one of claims 74 to 89, wherein the polypeptide comprises two to twenty independently selected mutant SPINK9 domains; and each mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula IV or SEQ ID NOs: 115-134.

91. The polypeptide of any one of claims 74 to 89, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain and a second mutant SPINK9 domain; and each of the first mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula IV or SEQ ID NO: 115-134.

92. The polypeptide of claim 91, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

93. The polypeptide of claim 91, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

94. The polypeptide of any one of claims 74 to 89, wherein the polypeptide comprises an independently selected first mutant SPINK9 domain, a second mutant SPINK9 domain, and a third mutant SPINK9 domain; and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, and the third mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula IV or SEQ ID NOs: 115-134.

95. The polypeptide of claim 94, wherein each of the mutant SPINK9 domains are identical.

96. The polypeptide of claim 94, wherein each of the mutant SPINK9 domains is different.

97. A polypeptide according to any one of claims 74 to 89, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, wherein the first polypeptide chain comprises a first mutant SPINK9 domain and a second mutant SPINK9 domain and the second polypeptide chain comprises a third mutant SPINK9 domain and a fourth mutant SPINK9 domain, and each of the first mutant SPINK9 domain, the second mutant SPINK9 domain, the third mutant SPINK9 domain and the fourth mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula IV or SEQ ID NO: 115-134.

98. The polypeptide of claim 97, wherein each of the mutant SPINK9 domains are identical.

99. The polypeptide of claim 97, wherein each of the mutant SPINK9 domains is different.

100. The polypeptide of any one of claims 90 to 99, wherein adjacent mutant SPINK9 domains are connected by direct fusion, a linker sequence, or any combination thereof.

101. The polypeptide of claim 101, wherein at least one mutant SPINK9 domain is connected to an adjacent mutant SPINK9 domain via a linker sequence.

102. The polypeptide of claim 101, wherein at least one mutant SPINK9 domain is linked to an adjacent mutant SPINK9 domain by direct fusion.

103. A polypeptide according to any one of claims 100 to 102, wherein the linker sequence comprises a cleavage site.

104. The polypeptide of claim 103, wherein the cleavage site is recognized by a subtilisin-like proprotein convertase.

105. The polypeptide of any one of claims 74 to 89, wherein the polypeptide comprises a first polypeptide chain and a second polypeptide chain connected via an Fc portion, wherein the first polypeptide chain contains a first mutant SPINK9 domain and the second polypeptide chain contains a second mutant SPINK9 domain, and each of the first mutant SPINK9 domain and the second mutant SPINK9 domain comprises an independently selected amino acid sequence of Formula IV or SEQ ID NO: 115-134.

106. The polypeptide of claim 105, wherein the first mutant SPINK9 domain is identical to the second mutant SPINK9 domain.

107. The polypeptide of claim 105, wherein the first mutant SPINK9 domain is not identical to the second mutant SPINK9 domain.

108. The polypeptide of any one of claims 1 to 107, further comprising a signal sequence.

109. The polypeptide of claim 108, wherein the signal sequence is linked to the amino (N) terminus of the polypeptide by direct fusion or through a linker sequence.

110. The polypeptide of claim 108, wherein the signal sequence is linked to the carboxyl (C) terminus of the polypeptide by direct fusion or through a linker sequence.

111. The polypeptide according to any one of claims 1 to 110, further comprising an immunoglobulin Fc region or a functional fragment thereof.

112. The polypeptide according to claim 111, wherein the immunoglobulin Fc region or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39-54.

113. The polypeptide according to claim 111, wherein the immunoglobulin Fc region or a functional fragment thereof comprises the amino acid sequence of formula II: Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro X1 Cys Pro Ala Pro X2 X3 X4Gly X5 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu X6 Ile X7 ArgX8 Pro Glu Val Thr Cys Val Val X9 Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr X 10 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val X 11 His X 12 Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys X 13 Leu Pro X 14 X 15 Ile Glu LysThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheTyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys LeuThr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val X 16 His GluAla Leu X 17 X 18 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys(SEQ ID NO:24); in: X1 is Pro or Ser; X2 is Glu or Pro; X3 and X4 are each independently Lue or Ala; X5 is Gly or Ala; X6 is Met or Tyr; X7 is Ser or Thr; X8 is Thr or Glu; X9 is Val or Glu; 10 is Asn or Ala; X 11 Leu or Asp; X 12 is Gln or His; X 13 is Ala or Gly; X 14 Ala or Ser; X 15 For Pro or Ser; X 16 Met or Leu; X 17 is His or Lys; and X 18 is Asn, Ser or Phe.

114. The polypeptide according to any one of claims 111 to 113, wherein the immunoglobulin Fc region or a functional fragment thereof is connected to the mutant SPINK9 domain by direct fusion or through a linker sequence.

115. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114.

116. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114, wherein amino acids 1-3 of SEQ ID NOs: 55-114 are deleted.

117. The polypeptide of any one of claims 1 to 3, wherein the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114.

118. The polypeptide of any one of claims 1 to 3, wherein the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-114, wherein amino acids 1-3 of SEQ ID NOs: 55-114 are deleted.

119. A nucleic acid encoding the polypeptide of any one of claims 1 to 118.

120. A vector comprising the nucleic acid of claim 119.

121. A host cell comprising the nucleic acid of claim 119 or the vector of claim 120.

122. A method for producing a polypeptide comprising at least one mutant SPINK9 domain, the method comprising expressing the polypeptide in a host cell according to claim 121, and The polypeptide is isolated.

123. A composition comprising the polypeptide of any one of claims 1 to 118, the nucleic acid of claim 119, the vector of claim 120 or the host cell of claim 121.

124. A pharmaceutical composition comprising the polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 118; and a pharmaceutically acceptable carrier.

125. A method of treating or preventing a disease or condition characterized by aberrant pancreatic vasodilator activity, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide according to any one of claims 1 to 105, or a pharmaceutically acceptable salt thereof.

126. The method of claim 125, wherein the disease or condition is characterized by aberrant pancreatic vasodilator-related peptidase 5 (KLK5) activity.

127. The method of claim 125 or 126, wherein the disease or condition is a skin disease.

128. The method of claim 125 or 126, wherein the disease or condition is Netherton Syndrome.

129. The method of claim 127, wherein the skin disease is eczema, atopic eczema, atopic dermatitis, autosomal recessive ichthyosis, ichthyosis-like erythroderma, psoriasis, UV-induced skin damage, rosacea, or a skin infection.

130. The method of claim 125 or 126, wherein the disease or condition is selected from the group consisting of: immune system hypersensitivity (atopy), hyper IgE syndrome, allergies (including allergies to food and airborne factors), asthma, allergic asthma, chronic inflammation, rhinitis, conjunctivitis, angioedema, eosinophilia, eosinophilic esophagitis, growth retardation, stunted development, trichorrhexis invaginata (TI), respiratory tract infections, systemic infections, and gastrointestinal disorders.

131. The method of claim 125 or 126, wherein the disease or condition is cancer.

132. The method of claim 131, wherein the cancer is selected from the group consisting of ovarian cancer, uterine cancer, colorectal cancer, bladder urothelial carcinoma, oral squamous cell carcinoma, breast cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, head and neck cancer glioma, glioblastoma multiforme, and neuroblastoma.

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