Modulation of neutrophil activation and neutrophil extracellular trap (NET) formation

Abstract

The present disclosure relates generally to methods of preventing or inhibiting neutrophil extracellular trap (NET) formation and methods of treating a disease or disorder associated with NET formation in a subject.

Description

MODULATION OF NEUTROPHIL ACTIVATION AND NEUTROPHIL EXTRACELLULAR TRAP (NET) FORMATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 506,712 filed 7 June 2023, the contents of which are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format as an XML file and hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 0398.0003-PCT_SL.xml and the size of the text file is 7,865 bytes.FIELD

[0003] The present disclosure relates generally to methods of preventing or inhibiting neutrophil extracellular trap (NET) formation and methods of treating a disease or disorder associated with NET formation in a subject.BACKGROUND

[0004] Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathophysiology and clinical manifestations of many different autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. It has been previously shown that inhibition of NET formation improves outcomes in animal models of autoimmune diseases (Knight et al., Ann. Rheum. Dis., 2015, 74:2199-2206; Gajendran et al., Scientific Reports, 2023, 13:3189; Heger et al., Life Sci. Alliance, 2023, 6(10):e202302055). Ways to prevent or inhibit NET formation, including IC-mediated NET formation, are therefore of considerable interest in treating a number of conditions in which the immune system is dysregulated, such as autoimmune diseases. However, to date, while there are approved agents that affect NET formation (e.g., disulfiram), there is no therapy specifically approved to stop orprevent NET formation. Accordingly, there remains a need for an effective therapy to prevent or inhibit NET formation.SUMMARY

[0005] The present disclosure is directed to methods of preventing or inhibiting neutrophil extracellular trap (NET) formation and methods of treating a disease or disorder associated with NET formation in a subject.

[0006] In one aspect, provided herein is a method of treating a disease or disorder associated with neutrophil extracellular' trap (NET) formation, said method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G. In some embodiments, the disease or disorder is a relapsing-remitting disease or disorder associated with NET formation, and the subject is in remission at the time of treatment. In some embodiments, administration of the molecule capable of inhibiting cathepsin G prolongs remission or prevents or delays onset of a relapse of the disease or disorder. In some embodiments, the disease or disorder is an immune complex driven autoimmune disease. In some embodiments, the disease or disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis. In other embodiments, the disease or disorder involves NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis.

[0007] In a further aspect, provided herein is a method of preventing or delaying onset of a relapse of a disease or disorder, wherein the relapse is mediated by neutrophil extracellular trap (NET) formation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G. In some embodiments, the disease or disorder multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

[0008] In some embodiments, the molecule capable of inhibiting cathepsin G used in any of the methods disclosed herein is a peptide, a polypeptide, or a non-peptide small molecule. In someembodiments, the molecule capable of inhibiting cathepsin G is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a nonstructured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a cathepsin G inhibitor I having a CAS registry number of CAS 429676-93-7. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide, an oxidized serpin B 1 polypeptide, or a serpin B 1 variant polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G is a protein that naturally inhibits cathepsin G. In some embodiments, the molecule capable of inhibiting cathepsin G is a protein that has been engineered to inhibit cathepsin G. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide, an oxidized serpin B 1 polypeptide, or a serpin B 1 variant polypeptide fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non- structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a prolinc-alaninc-scrinc polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, the serpin Bl polypeptide or oxidized serpin B 1 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin B 1 variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the amino acid substitution is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0009] In yet another aspect, provided herein is a method of preventing or reducing recurrence and / or severity of a bacterial infection in a subject suffering from episodic recurrent bouts of inflammation associated with neutrophil extracellular trap (NET) formation, said method comprising administering to the subject a therapeutically effective amount of a serpin B 1 variant polypeptide, wherein the serpin Bl variant polypeptide is oxidation-resistant. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is not immunocompromised. In some embodiments, the serpin Bl variant polypeptide is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a humantransferrin, or a non-structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine- serine polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, administration of the serpin B 1 variant polypeptide increases phagocytosis and boosts intracellular reactive oxygen species (ROS) production in the subject. In some embodiments, the serpin Bl variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the amino acid substitution is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the episodic recurrent bouts of inflammation are caused by a relapsing-remitting disease or disorder associated with NET formation, and the subject is in remission at the time of treatment. In some embodiments, administration of the serpin Bl variant polypeptide prolongs remission or prevents or delays onset of a relapse of the disease or disorder. In some embodiments, the relapsing -remitting disease or disorder associated with NET formation is multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), antineutrophilic cytoplasmic antibody (ANCA) associated vasculitis (AAV), small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis. In some embodiments, the subject is diagnosed with a disease or disorder involving NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and devices disclosed herein.[OOH] FIG. 1A-1B show inhibition of neutrophil serine proteases (NSPs) by serpin Bl variants. FIG. 1A: Complex formation of serpin Bl variants with NSPs; FIG. IB: Inhibition of NSP amidolytic activity on chromogenic substrates by serpin Bl variants. Panels: i) Purified proteins; ii) Purified proteins + hNE; iii) Purified proteins + hPR3; iv) Purified proteins + hCG. M = molecular weight markers (Genscript USA Inc., Page Master protein standard plus Mol weights: 120 kDa (not shown), 80 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 15 kDa, and 10kDa). The serpin Bl variants loaded onto lanes A-G of the SDS-PAGE gel correspond to the Codes provided in Table 1.

[0012] FIG. 2A-2F show inhibition of neutrophil extracellular trap (NET) formation by serpin Bl. Neutrophils were pretreated with various concentrations of serpin Bl variants then stimulated to undergo NET formation with immune complexes (IC, FIG. 2A-2B), PMA (FIG. 2C-2D), or A23187 (FIG. 2E-2F). Released NETs were assessed for DNA content (FIG. 2A, FIG. 2C, FIG. 2E) and DNA-bound peroxidase (MPO) activity (FIG. 2B, FIG. 2D, FIG. 2F) as described in Example 1. Results are presented as % DNA or peroxidase activity as compared to NET-inducing stimuli alone. DPI (25 pM) was used as a positive control. The results represent the mean + / - SD from more than four independent experiments. Statistical analyses were performed by paired t- test and compared pretreated and NET agonist stimulated cells with NET agonist stimulated cells only (* p<0.05, ** p<0.01 and *** p<0.001).

[0013] FIG. 3A-3B show inhibition of IC-mediated NET formation is dependent on the chymotrypsin-like inhibitory activity of serpin Bl. Neutrophils were pretreated with a fixed concentration of serpin B 1 variant, DPI, cathepsin G (Cat G) inhibitor, or sivelestat then stimulated to undergo NET formation with IC. Released NETs were assessed for DNA content (FIG. 3A) and DNA-bound peroxidase (MPO) activity (FIG. 3B) as described in Example 1. Results are presented as % DNA or peroxidase activity. The results represent the mean + / - SD from more than four independent experiments. Statistical analyses were performed by paired t-test comparing pretreated and IC stimulated cells with IC stimulated cells (* p<0.05, ** p<0.01 and *** p<0.001).

[0014] FIG. 4A-4D show serpin Bl variant C334A amplifies neutrophil activation marker expression and reactive oxygen species (ROS) production in response to activation by ICs. Neutrophils were pretreated with a fixed concentration of serpin B 1 variant, sivelestat, or DPI then stimulated to undergo NET formation with ICs. After 1 hour stimulation, cells were assessed for intracellular ROS generation (FIG. 4A), activated CD 11b (FIG. 4B), CD66b (FIG. 4C), and CD63 cell surface levels (FIG. 4D) by flow cytometry. The results represent the mean + / - SD from more than four independent experiments. Statistical analyses were performed by paired t- test comparing pretreated and IC-stimulated cells with IC-stimulated cells (* p<0.05, and ** p<0.01).

[0015] FIG. 5A-5B show serpin Bl enhances microbial phagocytosis. Neutrophils were incubated with fluorescently labeled immune complexes (FIG. 5A) or serum-opsonized bacteria(FIG. 5B) in the presence of various serpin Bl variants (5 pM) and assessed for uptake by flow cytometry as described in Example 1. The data are represented as the ratio as compared to no serpin Bl variant added. The results are from three independent experiments with mean + / - SD. Statistical analyses were performed by paired t-test comparing pretreated and IC-stimulated cells with IC-stimulated cells (* p<0.05, and ** p<0.01).

[0016] FIG. 6 depicts hypothetical role(s) of cathepsin G (hCG) in IC-mediated NET formation. In this model, immune complexes activate members of the SRC and Syk kinase families via FcgRIIIB and / or FcgRIIAl. Subsequent signaling pathways result in the rapid production of ROS and release of NSPs, including hCG, via degranulation. Surface bound hCG may then induce a secondary signal required for induction of NET formation via one or more of the pathways indicated including integrin (Mac-1) clustering, MMP activation, and subsequent Fey receptor shedding, and PAR, FPR, or TLR activation (via cytokine processing). Serpin Bl prevents NET formation via a mechanism involving binding to the neutrophil cell surface and direct inhibition of hCG.DETAILED DESCRIPTION

[0017] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as a limitation of the scope of the disclosure.Definitions

[0018] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0019] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described hereinand / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0020] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1%, including all values and subranges there between, from the specified value as such variations are appropriate to perform the disclosed methods and / or to make and use the disclosed devices. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0021] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but arc not limited to, oral administration, transdcrmal administration, administration by inhalation, nasal administration, enteral administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, intraurethral administration, transmucosal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, and subcutaneous administration. Administration can be continuous or intermittent. In some embodiments, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In some embodiments, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0022] The term “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B(optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0023] The term “associated with,” with regard to the relationship between a disease or disorder and neutrophil extracellular trap (NET) formation, indicates that the disease or disorder is at least in part resulted from NET formation. Diseases or disorders associated with NET formation may include, but are not limited to, IC mediated autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), antineutrophilic cytoplasmic antibody (ANCA) associated vasculitis (AAV), small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, and psoriasis. Other diseases associated with NET formation include, but are not limited to, asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis.

[0024] The term “at least” prior to a number or scries of numbers (c.g., “at least two”) is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0025] The term “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” as used herein refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and other similar factors. For example, it is possible to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, singledose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In some embodiments, a preparation can be administered in a “prophylactic ally effective amount;” that is, an amount effective for prevention of a disease or condition. Effective amount may be expressed, for example, in grams, milligrams or micrograms or as milligrams per kilogram of body weight (mg / kg). Alternatively, the effective amount can be expressed in the concentration of the active component (e.g., serpin Bl polypeptide of the present disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction and mixing ratio. Specifically, the term “effective amount” used in connection with serpin Bl polypeptides described herein refers to the quantity of serpin Bl polypeptides, which is sufficient to alleviate or ameliorate the symptoms associated with the NET formation in the subject. Persons having ordinary skills could calculate the human equivalent dose (HED) for the medicament (such as serpin Bl polypeptides of the present disclosure) based on the doses determined from animal models. For example, one may follow the guidance for industry published by US Food and Drug Administration (FDA) entitled “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” in estimating a maximum safe dosage for use in human subjects.

[0026] The term “identical” or “identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988). Typical methods to determine identityare designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0027] The term “inflammation” will be understood by those skilled in the ait to include any condition characterized by a localized protective response elicited by infection, and which is manifested by heat, swelling, pain, redness, dilation of blood vessels and / or increased blood flow, invasion of the affected area by white blood cells, loss of function, and / or any other symptoms known to be associated with the inflammatory condition. As used herein, the term “inflammation” refers to an accumulation, up-regulation and / or induction of pro-inflammatory agents (c.g., cells, cytokines, and chemokines) in a tissue and / or organ (e.g., the lung) of a subject.

[0028] The term “in need thereof” means that the subject has been identified or is suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods described herein, the subject can be in need thereof. In some embodiments, the subject in need thereof is a human suspected of having a disease or disorder associated with NET formation. In some embodiments, the subject in need thereof is a human diagnosed with a disease or disorder associated with NET formation. In some embodiments, the subject in need thereof is a human seeking treatment for a disease or disorder associated with NET formation. In some embodiments, the subject in need thereof is a human undergoing treatment for a disease or disorder associated with NET formation.

[0029] As used herein, the term “in some embodiments” or “in other embodiments” or any similar terminology refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0030] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0031] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like, are meant to refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.

[0032] The terms “protein” and “polypeptide” are used interchangeably herein and refer to a biological molecule consisting of one or more peptides, regardless of post-translational modification. Each peptide in a protein may be a subunit. The protein or polypeptide may be in a native or modified form and may exhibit a biological function or characteristics.

[0033] The terms “serpin Bl polypeptide,” “serpin Bl protein,” and “serpin Bl” are used interchangeably herein and refer to a polypeptide that is capable of inhibiting the serine protease activity of neutrophil cathepsin G and elastase. An “oxidation-resistant” serpin Bl polypeptide refers to a serpin B 1 polypeptide that is resistant to oxidation by a free radical, where the free radical is a reactive oxygen species, a reactive nitrogen species, or both.

[0034] The term “subject” as used herein can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. A “patient” refers to a subject afflicted with a disease or disorder, particularly humans, hospitalized or not. The term “patient” includes human and veterinary subjects. In some embodiments, the subject has been diagnosed with a need for treatment of an influenza virus infection prior to the administering step. In some embodiments, the subject has been diagnosed with having a disease or condition associated with an influenza virus infection prior to the administering step.

[0035] The term “treat,” “treated,” “treating,” or “treatment” refers to therapeutic treatment and / or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration ofthe condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.Activation of Neutrophils and formation of Neutrophil Extracellular Traps

[0036] Neutrophils are essential cells of the innate immune system capable of recognizing pathogens due to a broad spectrum of pattern recognition receptors, a wide array of Fc gamma receptors (FcgRs), and able to capture immune complexes (ICs) (Chen et al., Front Immunol., 2021, 12:775267; Wang et al., Front Immunol., 2019, 10:1958). Upon activation by ICs, whether targeting pathogen and / or self-antigen as in autoimmune conditions, cross-linking of FcgRs activates neutrophil effector functions, including phagocytosis, degranulation, reactive oxygen species (ROS) generation, and formation of neutrophil extracellular traps (NETs), a cell death process in which nuclear and cytosolic content is extruded in a web-like conformation to trap and eliminate the pathogen and / or self-antigen (Wang et al., Front Immunol., 2019, 10:1958; Granger et al., Front Immunol., 2019, 10:2824). However, exaggerated neutrophil activation, and NET formation in particular’, can be detrimental to human health, with NETs contributing to vascular damage, thrombosis, malignancy, inflammation, organ damage, and autoimmunity (Granger et al., Front Immunol., 2019, 10:2824; Thiam et al., Annu. Rev. Cell Dev. Biol., 2020, 36:191-218; Masucci et al., Front Immunol., 2020, 11:1749; Bach et al., Arthritis Rheumatol., 2020, 72:47-56; Kuley et al., Front Immunol., 2021, 12:785275; Michailidou et al., Arthritis Res. Then, 2022, 24: 160; Michailidou et al., Rheumatology, 2023, 62:2880-2886; Michailidou et al., Clin Immunol., 2023, 249:109274). Thus, it is useful to properly regulate IC-mediated neutrophil activation to ensure adequate clearance of the ICs, while preventing excessive neutrophil death and tissue damage. To this end, it has been shown that specifically targeting NET formation by inhibiting peptidyl arginine deaminase (PAD) activity led to reduced organ damage in mouse models of lupus, arthritis and cardiovascular disease (Knight et al., Ann. Rheum. Dis., 2015, 74:2199-2206; Gajendran et al., Scientific Reports, 2023, 13:3189; Heger et al., Life Sci. Alliance, 2023, 6(10):e202302055).

[0037] Neutrophil serine proteases (NSPs) released during neutrophil activation play an important role in shaping the immune response by activating cytokines and matrix metalloproteases and cleaving cell surface receptors, such as protease-activated receptors (PARs), to trigger receptor-mediated signaling pathways (Korkmaz et al., Pharmacological Reviews, 2010, 62:726-759). For example, cathepsin G (hCG) cleaves PAR-4 on platelets leading to intravascular thrombosis, tissue infarction, and release of platelet granule contents while neutrophil elastase (hNE) can cleave cell surface receptors on innate immune cells required for effective phagocytosis and removal of pathogens, ICs, and apoptotic neutrophils (Sambrano et al., J. Biol. Chem., 2000, 275:6819-6823; Ma et al., Am. J. Physiol. Lung Cell Mol. Physiol., 2021, 321:L555-L565). Alpha 1-antitrypsin (serpin Al, sAl, AAT) is the major human plasma protease inhibitor targeting NSPs. It has been tested as a potential anti-inflammatory agent for the treatment of autoimmune diseases including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus with positive results reported in each case (Song, Chronic Obstr. Pulm. Dis., 2018, 5:289-301). However, AAT docs not inhibit NET formation (Farley et al., J. Immunol., 2012, 189:4574-4581; Janciauskicnc et al., Frontiers Pharmacol., 2018, 9:341). Despite conflicting reports, the general consensus is that NSPs, in particular intracellular hNE, are involved in NET formation (Lelliott et al., Int. Immunol., 2020, 32:359-368; Wang et al., Int. Immunol., 2023, 114:109537). The current dogma is that ROS mediate translocation of hNE and MPO into the nucleus to facilitate de-condensation of chromatin together with PAD4 (Papayannopoulos et al., J. Cell Biol., 2010, 191:677-691; Tokuhiro et al., Frontiers Cell Dev. Biol., 2021, 9:718586). While many studies have demonstrated that inhibition of hNE in vitro and in vivo prevents NET formation, there are many other reports where elastase is not involved and, thus, other proteases may contribute to NET formation in these cases (Wang et al., Int. Immunol., 2023, 114:109537; Kolaczkowska et al., Nat. Communications, 2015, 6:6673; Martinod et al., I. Thromb. Haemost., 2016, 14:551-558; Tatsiy et al., Frontiers Immunol., 2021, 12:675315).Autoimmune Diseases

[0038] Autoimmune diseases are caused by a breakdown in self-tolerance, resulting in an immune response to self-antigens and eventual tissue damage. Autoimmune diseases effect nearly every organ, including the central nervous system (multiple sclerosis), endocrine system (type 1 diabetes), and skin (psoriasis), with diverse symptoms. One common feature of many autoimmune diseases is the occurrence of flares, also called relapses, which are episodes of increased diseaseseverity. For instance, patients with relapsing-remitting multiple sclerosis exhibit disease in which they experience serious clinical symptoms followed by remission and subsequent relapse. In these patients, remission may not fully recover function, resulting in some progression over time. Autoimmune diseases are thus often characterized by relapses and remissions in which symptoms appear and then subside. Other examples of such autoimmune diseases include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, and psoriasis.

[0039] Several factors can trigger a relapse, including infections, stress, and exposure to certain drugs or other environmental factors. It is believed that these triggering factors may disrupt the balance between immune effector cells and regulator)' cells, causing an enhanced autoimmune attack, such as exaggerated neutrophil activation and NET formation. The ability to prevent NET formation can thus preclude onset of relapse and prevent disease progression, at least for those whose relapses are associated with NET formation.Serpin Bl (Serine Proteinase Inhibitor Bl)

[0040] Serpin Bl, or serine protease inhibitor Bl, also known as leukocyte elastase inhibitor (LEI), is a multi-functional immunomodulatory serine protease inhibitor highly expressed intracellularly in myeloid cells of the innate immune system - notably neutrophils, monocytes and macrophages (Janoff et al., Lab. Invest., 1971, 25(6):565-571; Remold-O’Donnell, E., J. Exp. Med., 1985, 162:2142-2155; Remold-O’Donnell et al., J. Exp. Med., 1989, 169:1071-1086). It is also found in the circulation at low levels (Takebayashi et al., BMJ Open, Diabetes Research and Care, 2016, 4:e000274). Serpin Bl exhibits a number of protective anti-inflammatory roles in vivo.

[0041] Serpin Bl has been shown to restrict the NET formation induced by LPS, PMA, PAF and MIP-2 (cxcl2) and regulate the homeostatic expansion of pro-inflammatory IL-17+y5 and CD4+Thl7 phenotype T-cells (Farley et al., J. Immunol., 2012, 189:4574-4581; Zhao et al., J. Leukoc. Biol., 2014, 95:521-530). It has also been shown to directly restrict the activation of pro- inflammatory caspases and act as a growth factor for pancreatic P-islet cells (Choi et al., Nat. Immunol., 2019, 20(3):276-287; El Ouaamari et al., Cell Metabol., 2016, 23:1-12).

[0042] Serpin B 1 is a potent inhibitor of the major NSPs hNE, PR3 and hCG, but not NSP4 (Cooley et al., Biochemistry, 2001, 40:15762-15770; Perera et al., PNAS, 2012, 109:6229-6234). Unlike AAT, which utilizes Met358 as the Pl residue to inhibit NSPs, serpin Bl utilizes two independent adjacent amino acids to inhibit proteases - Phe343 (or F343) to inhibit chymotrypsin- like proteases (including chymotrypsin, neutrophil cathepsin G (catG), mast cell chymase, granzyme H (GmzH) and prostate specific antigen (PSA)) and Cys344 (or C344) to inhibit elastase-like proteases (including neutrophil proteinase 3 and several cysteine cathepsins including cathepsins L and S) (Cooley et al., Biochemistry, 2001, 40:15762-15770; Wang et al., J. Immunol., 2013, 190:1319-1330; Hou et al., J. Autoimmun., 2015, 65:56-63). Serpin Bl is a non-redundant critical survival factor for neutrophils during development that protects them against hPR3- mediated apoptosis and hCG-mediated necrosis (Loison et al., J. Clin. Invest., 2014, 124:4445- 4458; Burgener et al., Cell Reports, 2019, 27:3646-3656.e3645; Loison et al., Inflamm. Cell Signal, 2014, l:e462). Neutrophils lacking serpin Bl are also predisposed to enhanced NET formation induced by PMA, PAF, MIP-2 and LPS (Farley et al., J. Immunol., 2012, 189:4574- 4581). Further, exogenous recombinant human serpin Bl (rhsBl) can restrict NET formation when added to wild-type neutrophils prior to treatment with these activating agents. However, its mechanism of action is unknown and it is unclear whether inhibition by rhsB 1 is restricted to these agents or occurs broadly upon NET formation induced by other biologically relevant agents.

[0043] The structurally exposed C344 in the wild-type serpin Bl, however, renders it susceptible to oxidation by free radicals, leading to redox-mediated post-translational modifications that result in a significant reduction or a complete loss of elastase inhibitory activity (Pemberton, P. A., Protein Expr. Purif., 2020, 170:105595; WO 2021 / 022212). Thus, the neutrophil (and pancreatic) elastase inhibitory activity of the wild-type serpin Bl is dependent on the reduced form of C344. The term “free radical” or “radical,” or “reactive free radical species” typically refers to a molecule with an unpaired electron and capable of high reactivity, such as reactive oxygen species (ROS) or reactive nitrogen species (RNS). Radicals have extremely high chemical reactivity and when generated in excess or not appropriately controlled, may inflict damage upon cells. There are numerous potential endogenous and exogenous sources of free radicals that could oxidize C344 in exogenously administered wild-type serpin Bl, such as peroxidase produced hypohalous acids and cigarette smoke. However, it is important to note that not all free radicals can oxidize C344. For example, hydrogen peroxide - the most commonproduct of metabolic respiration - cannot, while the more potent hypohalous acids (e.g., hypochlorous acid) or RNS (e.g., peroxynitrite) can. Preventing this potential oxidation and maintaining C334 in a reduced state may allow exogenously administered serpin Bl to retain its innate elastase inhibitory activity and enhance its in vitro and in vivo efficacy. This can be achieved by, for instance, co-administering the serpin Bl polypeptide with a reducing agent that prevents oxidation of C334, such as N-acetylcysteine (NAC), or using an oxidation-resistant serpin Bl polypeptide that possesses neutrophil or pancreatic elastase inhibitory activity that is resistant to oxidation by a free radical.

[0044] Human serpin Bl was first identified in 1971 as an elastase inhibitory activity (EIA) present in macrophages (Janoff et al., Lab. Invest., 1971, 25(6):565-571). It has a molecular weight of approximately 42 kDa with the amino acid sequence set forth below (C334 is bold and italicized):MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTA AQLSKTFHFNTVEEVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFL PEFLVSTQKTYGADLASVDFQHASEDARKTINQWVKGQTEGKIPELLASG MVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDRKTVKMM YQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQ LTLEKLHEWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSS KADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFCMLMPEE NFTADHPFLFFIRHNSSGSILFLGRFSSP (GenBank Accession No. NP_109591; SEQ ID NO: 1).

[0045] Human serpin Bl variants that are oxidation resistant were previously disclosed in, for instance, Pemberton, P. A., Protein Expr. Purif., 2020, 170:105595 and WO2021 / 022212, both incorporated herein by reference in their entirety. Such oxidation-resistant human serpin Bl variants may include, but are not limited to, human serpin Bl variants having a substitution at C344, such as a C344G variant, a C344A variant, a C344V variant, or a C344S variant, with their corresponding amino acid sequences set forth below (the substitution at residue C344 is bold and italicized), although it should be noted that C344S variant is a poor inhibitor of porcine pancreatic elastase as disclosed in WO2021 / 022212:Human serpin B 1 C344A variant:MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHFNTVEEVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHASEDARKTINQWVKGQTEGKIPELLASGMVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDRKTVKMMYQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQLTLEKLHEWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFAMLMPEENFTADHPFLFFIRHNSSGSILFLGRFSSP (SEQ ID NO: 2).Human serpin Bl C344G variant:MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHFNTVEEVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHASEDARKTINQWVKGQTEGKIPELLASGMVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDRKTVKMMYQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQLTLEKLHEWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFGMLMPEENFTADHPFLFFIRHNSSGSILFLGRFSSP (SEQ ID NO: 3).Human serpin B 1 C344V variant:MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHFNTVEEVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHASEDARKTINQWVKGQTEGKIPELLASGMVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDRKTVKMMYQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQLTLEKLHEWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFVMLMPEENFTADHPFLFFIRHNSSGSILFLGRFSSP (SEQ ID NO: 4).Human serpin Bl C344S variant:MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHFNTVEEVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHASEDARKTINQWVKGQTEGKIPELLASGMVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDRKTVKMMYQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQ LTLEKLHEWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSS KADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFSMLMPEE NFTADHPFLFFIRHNSSGSILFLGRFSSP (SEQ ID NO: 5).Methods of Treatment

[0046] The present disclosure is based, in part, on the discovery that rhsB 1 but not AAT can prevent neutrophil activation and NET formation mediated by immune complexes and other inducing agents such as the calcium ionophore A23187 and PKC activator phorbol myristate acetate (PMA). This effect is dependent on its ability to inhibit cathepsin G as follow-up studies with a specific inhibitor of cathepsin G gave similar results. This discovery is unexpected and surprising because cathepsin G is a well-known contributor to autoimmune diseases but, to the knowledge of the inventors, this is the first time cathepsin G has been implicated as a potential driver of NET formation mediated by immune complexes and other agonists, and this is the first demonstration that blocking cathepsin G may potentially prevent NET formation. Accordingly, in one aspect, provided herein is a method of preventing or inhibiting NET formation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G. In some embodiments, the subject is diagnosed with a disease or disorder involving NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis. In other embodiments, the subject is diagnosed with a relapsing-remitting disease or disorder associated with NET formation, and the subject is in remission at the time of administration. In some embodiments, administration of the molecule capable of inhibiting cathepsin G prolongs the remission or prevents or delays onset of a relapse of the disease or disorder. In some embodiments, the relapsing-remitting disease or disorder associated with NET formation is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

[0047] As certain diseases and disorders are caused by dysregulated neutrophil activation and NET formation, such as autoimmune diseases, the ability to prevent neutrophil activation and NET formation can be an effective way to treat such diseases and disorders. Accordingly, also provided herein is a method of treating a disease or disorder associated with NET formation, said methodcomprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G. In some embodiments, the disease or disorder involves NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis. In some embodiments, the disease or disorder is a relapsing-remitting disease or disorder associated with NET formation and the subject is in remission at the time of treatment. In some embodiments, administration of the molecule capable of inhibiting cathepsin G prolongs remission or prevents or delays onset of a relapse of the disease or disorder. In some embodiments, the disease or disorder is an immune complex driven autoimmune disease. In some embodiments, the disease or disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

[0048] In relapsing-remitting types of diseases or disorders, particularly those in which the relapse is mediated by exaggerated neutrophil activation and NET formation, the ability to prevent NET formation can thus intervene onset of a relapse. Accordingly, further provided herein is a method of preventing or delaying onset of a relapse of a disease or disorder, wherein the relapse is mediated by NET formation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G. In some embodiments, the disease or disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

[0049] Cathepsin G belongs to the neutrophil serine proteases superfamily, known for its function in killing pathogens. Cathepsin G is present in the azurophilic granules of neutrophil leukocytes and other myeloid cells, such as B cells, primary human monocytes, myeloid dendritic cells, plasmacytoid dendritic cells, and murine microglia. Recent studies show that cathepsin G is also present in NETs and human urine exosomes. Studies indicate that cathepsin G has important effects on inflammation and immune reactions and may be a key factor in the pathogenesis of some autoimmune diseases. For a review of cathepsin G and its role in inflammation and autoimmune diseases, see for example, Gao et al., Arch. Rheumatol., 2018, 33(4):498-504.

[0050] Cathepsin G can be inhibited by [2-[3-[[(l-benzoyl-4-piperidinyl)methylamino] carbonyl]-2-naphthalenyl]-l-(l-naphthalenyl)-2-oxoethyl]-phosphonic acid (KPA) (Son et al., Dermatology, 2012, 224(4):352-360), Caesalpinia echinata elastase inhibitor (Cruz-Silva et al., Phytochemistry, 2013, 96:235-243), and A-arylacyl O-sulfonated aminoglycosides (Craciun et al., Glycobiology, 2016, 26(7):701-709). Caesalpinia echinata elastase inhibitor and / V-arylacyl O- sulfonated aminoglycosides can inhibit human neutrophil elastase, cathepsin G, and proteinase 3 (Cruz-Silva et al., Phytochemistry, 2013, 96:235-243; Craciun et al., Glycobiology, 2016, 26(7):701-709), while KPA has the strongest potency and selectivity against cathepsin G (Son et al., Dermatology, 2012, 224(4):352-360). KPA, now commonly known as cathepsin G inhibitor I, has a CAS registry number of CAS 429676-93-7 and the following chemical structure:Cathepsin G inhibitor I (CAS 429676-93-7).

[0051] In addition to cathepsin G inhibitor I, as demonstrated in Example 1, human serpin Bl, including wild-type serpin B 1 and oxidized serpin B 1 lacking hNE and PR3 inhibitory activity, as well as variants thereof, can also selectively inhibit cathepsin G. As used herein, an “oxidized serpin Bl polypeptide” refers to a serpin Bl polypeptide oxidized at C344.

[0052] Accordingly, in some embodiments, the molecule capable of inhibiting cathepsin G and useful for practicing the methods of the present disclosure can be a peptide, a polypeptide, or a non-peptide small molecule. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a non-peptide small molecule, such as Cathepsin G inhibitor I. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is cathepsin G inhibitor I having a CAS registry number of CAS 429676-93-7. In some embodiments, the molecule capable of inhibiting cathepsin G comprises, or is, a polypeptide, such as a serpin Bl polypeptide, an oxidized serpin Bl polypeptide, or a serpin Bl variant polypeptide. In some embodiments, themolecule capable of inhibiting cathepsin G comprises, or is, a serpin Bl polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises, or is, an oxidized serpin Bl. In some embodiments, the molecule capable of inhibiting cathepsin G comprises, or is, a serpin B 1 variant polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G is a protein that naturally inhibits cathepsin G. In some embodiments, the molecule capable of inhibiting cathepsin G is a protein that has been engineered to inhibit cathepsin G.

[0053] It has been shown that protein fusion technologies, such as Fc fusion proteins, fusion to human serum albumin (HS A), fusion to carboxy-terminal peptide, and other polypeptide fusion approaches, can be used to improve the pharmacokinetic properties of biologies, such as extending half-life. See e.g., Strohl, BioDrugs, 2015, 29(4):215-239. For instance, conjugation to either linear or branched-chain monomethoxy poly-ethylene glycol (PEG) (i.e., PEGylation) has been used widely as a means to lengthen the half-life of proteins. Fusion to human serum albumin (HSA), fusion to the constant fragment (Fc) domain of a human immunoglobulin (Ig)G, or fusion to non- structured, inert polypeptides, such as XTEN (also known as recombinant PEG or “rPEG”; Schellenberger et al., Nat. Biotechnol., 2009, 27: 1186-1190), a homo-amino acid polymer (HAP, HAPylation; Schlapschy et al., Protein Eng. Des. Sei., 2007, 20:273-284.), a proline-alanine-serine polymer (PAS, PASylation; Schlapschy et al., Protein Eng. Des. Sei., 2013, 26:489-501.), or an elastin-like peptide (ELP, ELPylation; Floss et al., Trends Biotechnol., 2010, 28:37-45) have also been widely used.

[0054] Accordingly, in some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure is fused to human HSA. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to a human IgG Fc domain. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to a human transferrin.

[0055] In some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure is fused to a non- structured, inert polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to XTEN. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to a homo-amino acid polymer. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to a proline-alanine-serine polymer. In some embodiments, the molecule capable of inhibiting cathepsin G is fused to an elastin-like peptide. In some embodiments, the molecule capable ofinhibiting cathepsin G is conjugated to a linear or branched-chain monomethoxy poly-ethylene glycol (PEG), i.e., PEGylated).

[0056] In some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure comprises or is a serpin B 1 polypeptide fused to a human IgG Fc domain. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to a human HSA. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to a human transferrin. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to a non-structured, inert polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide fused to XTEN. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to a homo-amino acid polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to a proline- alanine- serine polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl polypeptide fused to an elastin-like peptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide conjugated to a linear or branched-chain monomethoxy poly-ethylene glycol (PEG), i.e., PEGylated).

[0057] In some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure comprises or is an oxidized serpin B 1 polypeptide fused to a human IgG Fc domain. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to a human HSA. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin B 1 polypeptide fused to a human transferrin. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to a non-structured, inert polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to XTEN. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to a homo-amino acid polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to a proline- alanine- serine polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is an oxidized serpin Bl polypeptide fused to an elastin-like peptide. In some embodiments, the molecule capable ofinhibiting cathepsin G comprises or is an oxidized serpin B 1 polypeptide conjugated to a linear or branched-chain monomethoxy poly-ethylene glycol (PEG), i.e., PEGylated).

[0058] In some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure comprises or is a serpin B 1 variant polypeptide fused to a human IgG Fc domain. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to a human HSA. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 variant polypeptide fused to a human transferrin. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to a non-structured, inert polypeptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to XTEN. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to a homo-amino acid polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to a prolinc-alaninc-scrinc polymer. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide fused to an elastin-like peptide. In some embodiments, the molecule capable of inhibiting cathepsin G comprises or is a serpin Bl variant polypeptide conjugated to a linear or branched-chain monomethoxy poly-ethylene glycol (PEG), i.e., PEGylated).

[0059] As described above, the wild-type human serpin Bl comprises the amino acid sequence of SEQ ID NO: 1 and exemplary human serpin B 1 variant polypeptides include, but arc not limited to, human serpin B 1 C344G variant, C344A variant, C344V variant, and C344S variant with their corresponding amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. Accordingly, in some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure comprises or is a serpin Bl polypeptide or an oxidized serpin Bl polypeptide comprising an amino acid sequence that is at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, including all values and subranges therebetween, identical to SEQ ID NO: 1, optionally fused to a human IgG Fc domain, a human HSA, a human transferrin, or a non-structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine- serine polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, the serpin B 1 polypeptide or oxidized serpin B 1 polypeptide comprises an amino acid sequencethat is at least 90% identical to SEQ ID NO: 1. In some embodiments, the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0060] In some embodiments, the molecule capable of inhibiting cathepsin G useful for the methods of the present disclosure comprises, or is, a serpin B 1 variant polypeptide comprising an amino acid sequence that is at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, including all values and subranges therebetween, identical to SEQ ID NO: 1 and comprising an amino acid substitution at residue 344, optionally fused to a human IgG Fc domain, a human HSA, a human transferrin, or a non-structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline- alanine-serine polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, the serpin B 1 variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the serpin Bl variant polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the serpin B 1 variant polypeptide comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1 except for the amino acid residue at position 344.

[0061] In some embodiments, the amino acid substitution at residue 344 is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344A as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344G as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344V as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0062] The present disclosure is also based, in part, on the discovery that an oxidation-resistant rhsBl was more effective than wild-type rhsBl or AAT at maintaining elevated levels of cell surface markers required for neutrophil adhesion, migration and phagocytosis and at boosting intracellular ROS production and microbial phagocytosis. This discovery is unexpected and surprising because it implies that the oxidation-resistant form of serpin Bl, but not an oxidized form or the wild-type form, can impact additional neutrophil effector functions such as microbial killing. Because immunocompromised patients do not have the ability to respond normally to an infection, such as a bacterial infection, due to an impaired or weakened immune system, they often suffer from episodic recurrent bouts of inflammation due to recurrence of an infection. The ability to prevent NET formation and boost microbial killing makes the oxidation-resistant form of serpin B 1 particularly useful in preventing or reducing recurrence and / or severity of a bacterial infection in an immunocompromised subject who suffers from episodic recurrent bouts of inflammation associated with NET formation. Accordingly, in another aspect, provided herein is a method of preventing or reducing recurrence and / or severity of a bacterial infection in a subject suffering from episodic recurrent bouts of inflammation associated with NET formation, said method comprising administering to the subject a therapeutically effective amount of a serpin B 1 variant polypeptide, wherein the serpin Bl variant polypeptide is oxidation-resistant. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is not immunocompromised. In some embodiments, the oxidation-resistant serpin Bl variant polypeptide useful for such methods is fused to a human IgG Fc domain, a human HSA, a human transferrin, or a non-structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine- serine polymer, or an elastin-like peptide, or is PEGylated. In some embodiments, administration of the serpin Bl variant polypeptide, or fusion protein thereof, increases phagocytosis and boosts intracellular reactive oxygen species (ROS) production in the subject. In some embodiments, the subject is diagnosed with a disease or disorder involving NET formation, such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, or bronchiectasis. In some embodiments, the episodic recurrent bouts of inflammation associated with NET formation is caused by a relapsing-remitting disease or disorder associated with NET formation, and the subject is in remission at the time of treatment. In such embodiments, administration of the serpin Bl variant polypeptide, or fusion protein thereof, prolongs remission or prevents or delays onset of a relapse of the disease or disorder. In some embodiments, the relapsing-remittingdisease or disorder associated with NET formation is multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), antineutrophilic cytoplasmic antibody (ANCA) associated vasculitis (AAV), small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

[0063] In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises an amino acid sequence that is at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, including all values and subranges therebetween, identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1 except for the amino acid residue at position 344.

[0064] In some embodiments, the amino acid substitution at residue 344 is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344A as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344G as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344V as compared to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is C344S as compared to the sequence of SEQ ID NO: 1. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the serpin Bl variant polypeptide that is oxidation-resistant comprises the amino acid sequence of SEQ ID NO: 5.

[0065] The amount of the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the amountof the serpin Bl variant polypeptide that is oxidation-resistant used for administration in any of the methods disclosed herein may vary, but it should be in an amount that is therapeutically effective. In some embodiments, the therapeutically effective amount is from about 1 mg / kg to about 1,000 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 1 mg / kg to about 500 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 1 mg / kg to about 200 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 1 mg / kg to about 100 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 2.5 mg / kg to about 200 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 5 mg / kg to about 150 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 7.5 mg / kg to about 100 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 8 mg / kg to about 75 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is from about 10 mg / kg to about 100 mg / kg body weight per dose. In some embodiments, the therapeutically effective amount is about 1, 2.5, 5, 7.5, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg / kg body weight per dose.

[0066] To produce a therapeutic effect on the subject, the total dosage for a full course of treatment is from about 4 mg / kg to about 4,000 mg / kg body weight. In some embodiments, the therapeutically effective amount is from about 4 mg / kg to about 2,000 mg / kg body weight for a full course of treatment. In some embodiments, the therapeutically effective amount is from about 4 mg / kg to about 800 mg / kg body weight for a full course of treatment. In some embodiments, the therapeutically effective amount is from about 4 mg / kg to about 400 mg / kg body weight for a full course of treatment. In some embodiments, the therapeutically effective amount is from about 8 mg / kg to about 300 mg / kg body weight for a full course of treatment. In some embodiments, the therapeutically effective amount is from 30 mg / kg to about 300 mg / kg body weight for a full course of treatment. In some embodiments, the therapeutically effective amount is about 4, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 mg / kg body weight for a full course of treatment.

[0067] The molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin Bl polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant can be administered on a regimen of 1 to 4 times per day, once or twice per week, or once or twice per month. The dosing regimen can be adjusted to provide the optimal therapeutic response. It should be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the subject. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease undergoing therapy.

[0068] The molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or a variant thereof, or the serpin B 1 variant polypeptide that is oxidation-resistant used in any of the methods disclosed herein may be administrated by any route that is suitable for providing a pharmaceutical preparation to a subject. Such methods include, but arc not limited to, oral administration, transdcrmal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, intraurethral administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. In some embodiments, the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant is administered to the subject by a route selected from the group consisting of oral, enteral, nasal, topical, transmucosal, and parenteral administration. In some embodiments, the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the serpin B 1 variant polypeptide that is oxidation-resistant is administered parentally to the subject by intramuscular, intravenous, or intraperitoneal injection. Administration can be continuous or intermittent.

[0069] The molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin Bl polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant used in any of the methods disclosed herein may beadministered as a stand-alone active ingredient. Alternatively, and more often in practice, a pharmaceutically acceptable carrier can be used together with the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant to form a pharmaceutical composition for administration. Such pharmaceutical compositions, include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration. Such pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin Bl polypeptide (wild-type or oxidized form) or variant thereof, or the serpin B 1 variant polypeptide that is oxidation-resistant, or a pharmaceutical composition comprising the same can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds usually used for the treatment of diseases or disorders disclosed herein.

[0070] As the active ingredient, the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant can be combined with the pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, in some embodiments, the pharmaceutical compositions of the disclosure can be presented as discrete units suitable for oral administration, such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. In some embodiments, the pharmaceutical compositions of the disclosure can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water- in-oil liquid emulsion. In addition to the common dosage forms set out above, the pharmaceutical compositions of the disclosure can also be administered by controlled release means and / or delivery devices. The pharmaceutical compositions of the disclosure can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier. In general, the pharmaceutical compositions are prepared by uniformly admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0071] The pharmaceutically acceptable carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0072] In preparing the pharmaceutical compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0073] A tablet containing the pharmaceutic composition of the disclosure can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0074] Pharmaceutical compositions of the disclosure suitable for parenteral administration can be prepared as solutions or suspensions of the active ingredient in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0075] Pharmaceutical compositions of the disclosure suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringeability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0076] Pharmaceutical compositions of the disclosure can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin B 1 polypeptide (wild-type or oxidized form) or variant thereof, or the serpin B 1 variant polypeptide that is oxidation-resistant with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0077] In addition to the aforementioned carrier ingredients, the pharmaceutical compositions described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other ingredients can be included to render the pharmaceutical composition isotonic with the blood of the intended recipient. Compositions containing the molecule capable of inhibiting cathepsin G, such as cathepsin G inhibitor I or serpin Bl polypeptide (wild-type or oxidized form) or variant thereof, or the serpin Bl variant polypeptide that is oxidation-resistant according to the disclosure can also be prepared in powder or liquid concentrate form.

[0078] The following Examples are provided to elucidate certain aspects of the present disclosure and to aid those of skilled in the ail in practicing the disclosed subject matter. These Examples are in no way to be considered to limit the scope of the disclosure in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description provided herein, utilize the present disclosure to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.EXAMPLESExample 1. Serpin Bl Modulation of Immune Complex-Mediated Neutrophil Activation and NET Formation Requires its Chymotrypsin-like Inhibitory Activity.

[0079] Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Inhibition of NET formation improvesoutcomes in animal models of autoimmune diseases. Serpin Bl (sBl) is a major intracellular neutrophil proteinase inhibitor that can regulate NET formation both intracellularly and when administered prophy tactically to activated isolated neutrophils as a recombinant protein (rhsBl). However, its mechanism(s) of action are currently unknown as is its ability to block IC-mediated NET formation. This study was designed to determine the effect of rhsB 1 on IC-mediated NET formation, neutrophil activation and phagocytic activity and explore the molecular basis of any observed inhibition. We engineered or post-translationally modified, rhsBl proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with IC’s derived from patients with SLE, PM A or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were performed with rhsBl pretreated and IC-activated neutrophils. We found rhsBl dose-dependently inhibited NET formation mediated by all three agents and this activity to be critically dependent on its chymotrypsin-like protease inhibitory function. Follow-up studies with a specific cathepsin G inhibitor identified it as the likely protease inhibited by rhsBl that triggers NET formation. Only one rhsBl variant tested (C344A) increased surface levels of neutrophil adhesion / activation markers on IC-activated neutrophils and boosted intracellular ROS production. Subsequently we found that rhsBl, like alpha- 1 -antitrypsin, can enhance complement- mediated neutrophil phagocytosis of opsonized bacteria but neither protein enhanced clearance of ICs.

[0080] In conclusion we have identified a novel mechanism of action involving Cathepsin G by which exogenously administered rhsBl inhibits IC, PMA and A23187 mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB 1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysomes.1. Experimental procedures i. Design, expression and purification of serpin Bl variants

[0081] Recombinant human serpin Bl (rhsBl) and several engineered variants possessing altered protease inhibitory activities were produced in and purified from the yeast S. cerevisiae as previously described (Pemberton, 2020, Protein expression and purification, 170:105595) and summarized in Table 1. Several properties of wild-type (C344, code “D”) and an oxidationresistant valiant (C344A, code “C”) of rhsB 1 have been described previously (Cooley et al., 2001, Biochemistry, 40:15762-15770; Pemberton, 2020, Protein expression and purification, 170:105595; Cooley et al., 2001, Protein expression and purification, 2001, 14:38-44). The additional valiants created used either the wild-type or C344A sBl gene expression constructs as templates for site-directed mutagenesis. All genetic manipulations and confirmation of the final construct sequences were performed at Genscript USA Inc. (Piscataway, NJ). The variants were T331R (wild-type template, code “F”) and F343D, C344A, M345R (C344A template, code “G”). As has been described for other serpins, the replacement of T with R at amino acid residue 331 (T331R) within the hinge region of a serpin is predicted to restrict reactive center loop (RCL) mobility (“insertion blocker”) and prevent effective complex formation with a target protease. This conformational change is required for effective protease inhibition and variants with this mutation are predicted to be devoid of protease inhibitory activity (Lawrence et al., 1994, The Journal of biological chemistry, 269:27657-27662). The F343D, C344A, and M345R variants have been described elsewhere as a specific inhibitor of hPR3 that lacks any residual chymotrypsin-like inhibitory activity but retains elastase inhibitory activity (Jegot et al., 2011, FASEB J., 25:3019-3031). All preparations of variants and post-translationally modified forms of rhsBl were tested for endotoxin content using the gel clot method (Genscript, NJ) and if necessary treated using the phase separation method to reduce endotoxin content to levels acceptable for cell based assays (Aida et al., 1990, Journal of immunological methods, 132:191-195).Table 1. Neutrophil serine protease inhibitory properties of serpin Bl variants.1: hNE: human neutrophil elastase; hPR3: human proteinase 3; hCG: human cathepsin G2: experimental3: RCL: reactive center loop ii. Preparation of post-translationally-modified forms of sBl

[0082] Purified wild-type rhsBl was oxidized with N-chlorosuccinimide to create rhsBlOxR (code “A”) as previously described (Pemberton, 2020, Protein expression and purification, 170:105595) and re-purified by chromatography on a 5-mL HiTrap Q HP column cartridge. Oxidized rhsB 1 cannot inhibit either neutrophil or pancreatic elastase. Instead, it is rapidly cleaved within the reactive center loop to yield a non-inhibitory protein (rhsBl-CL, code “B”). RhsBl- CL was made from rhsBlOxR using pancreatic elastase and re-purified by anion-exchange chromatography .Hi. Protease inhibitory activity of serpin Bl variants

[0083] The inhibitory activity of rhsB 1 and its variants was tested against the major neutrophil serine proteases (NSPs), hCG and hPR3 (Athens Research and Technology, Athens, GA). Proteins were analyzed for their ability to inhibit these enzymes by two methods, the first is the formation of classical SDS and heat-stable complexes and the second is the titrimetric analysis of inhibition of NSP amidolytic activity against select chromogenic substrates. For the first method, aliquots (2 pL) of hNE, hPR3 and hCG (23.4 pM each) were mixed with each sBl protein at a 1:1 molar ratio in a fixed volume of 20 pL PBS, pH 7.4 for 15 minutes. Residual enzyme activity was stopped by the addition of a protease inhibitor mix (Sigma Aldrich, St Louis, MO) and samples analyzed by SDS-PAGE after the addition of sample buffer (reducing) and heating. For the second method, each sBl protein was incubated with each enzyme at molar ratios varying from 0.5:1 to 4:1 inhibitor-to-enzyme in a 200 pL volume of PBS, pH 7.4 in a microtiter plate for 5 minutes. The concentration of hNE was 100 nM and residual enzyme activity (Vmax) measured via release of p-nitroaniline (405 nm) from MeO-Succ AAPV-pNA (Bachem Americas, Torrance, CA). hCG was 500 nM and residual enzyme activity (Vmax) measured via release of p-nitroaniline (405 nm) from Succ AAPF-pNA (Bachem Americas, Torrance, CA). hPR3 was 200 nM and residual enzyme activity (Vmax) measured via release of p-nitroanilinc (405 nm) from MeO-Succ AAPV- pNA. All synthetic substrate final concentrations were 1 mM.iv. NET formation assays

[0084] Human neutrophils were isolated by Polymorphprep™ (Axis-Shield, Dundee, UK) as previously described (Lood et al., 2016, Nat. Med., 22:146-153). Red blood cells were lysed with RBC lysis buffer (BioLegend, San Diego, CA). Neutrophils were resuspended in serum- free RPML1640 medium (Life Technologies, Waltham, MA) for in vitro assays. Prior observations had determined that rhsB 1 only inhibited NET formation when added prior to the addition of the NET stimulating agent (Farley et ah, Journal of immunology, 189:4574-4581). Hence, after 20 minutes settling down in poly-L-lysine-coated tissue culture plates, neutrophils (106cells / mL) were pre-incubated for another 60 minutes in the presence of rhsBl, an rhsBl variant, or other reagent at the indicated concentrations prior to addition of RNP-IgG immune complexes (ICs) prepared as described (Lood et ah, J. Exp. Med., 2017, 214:2103-2119; Wang et ah, Lupus Science & Medicine, 2022, 9:e000709) (IgG; purified from SLE patients with high titer anti- ribonucleoprotein [RNP] antibodies mixed with SmRNP [Arotec Diagnostics, Lower Hutt, Wellington, New Zealand], and used at a final concentration of 10 pg / mL). The operator was blinded to which inhibitors were used. In some experiments, neutrophils were activated with PMA (20 nM) or A23187 (25 pM). After 4 hours of incubation, NETs were washed in PBS once, and detached using micrococcal nuclease (0.3 U / mL; Thermo Fisher Scientific, Waltham, MA), diluted in nuclease buffer containing 10 mM Tris-HCl, pH 7.5, 10 mM MgCk, 2 mM CaCh, and 50 mM NaCl. Detached NETs were quantified by analyzing Sytox Green™ (Life Technologies, Carlsbad, CA) intensity and peroxidase activity (TMB) by plate reader (Synergy 2; BioTek, Winooski, VT). v. Neutrophil activation assays

[0085] Neutrophils were pre-incubated with rhsB 1 or a variant, then activated with RNP-IC (10 pg / mL), as above, for 3 hours, and analyzed for surface levels of CD66b (#305106), CD l ib (#301406; recognizing the activated form of CDl lb), and CD63 (#353008; all monoclonal antibodies diluted 1 / 100, BioLegend, San Diego, CA) by flow cytometry. Data were analyzed by FlowJo (Tree Star, Ashland, OR). vi. Reactive oxygen species (ROS) assays

[0086] Neutrophils were isolated and suspended in RPMI before being put on ice for 30 minutes. Neutrophils (IxlO6cells / mL) were then transferred into a 96-well U-bottom plate in the presence of different rhsBl variants (5 pM) or diphenyleneiodonium (DPI, 25 pM, Sigma, St. Louis, MO) for a 1-hour incubation at 37°C. R848 (2.5 pg / mL, InvivoGen, Carlsbad, CA) orRNP-IC (10 pg / mL) were used to stimulate neutrophils for an additional hour at 37 °C. During the last half hour of incubation, dihydrorhodamine 123 (DHR-123, Thermo Fisher Scientific, Waltham, MA) was added to each well (0.5 pM). Cells were analyzed by flow cytometry immediately for ROS production at the end of incubation. vii. Neutrophil phagocytosis of fluorescence-labeled immune complexes

[0087] Purified anti-RNP SLE IgG was labeled with Alexa Fluor™ 488 Antibody Labeling Kit (A20181, Thermo Fisher Scientific, Waltham, MA) at appropriate concentration according to the manufacturer’s instructions prior to incubation with SmRNP to make Alexa Fluor™ 488- labeled immune complexes. Isolated neutrophils (1 x 106cells / mL) were incubated with or without different rhsBl compounds at 5 pM (B, C, D, and E) for 1 hour prior to the addition of Alexa Fluor™ 488-labeled immune complexes for another one hour. At the end of incubation, the neutrophils were placed on ice to stop further phagocytosis. After washing, neutrophil phagocytosis of fluorescence-labeled immune complexes was determined with flow cytometry by assessing the mean fluorescence intensity (MFI) of Alexa Fluor™ 488. viii. Neutrophil phagocytosis of complement-opsonized staphylococcus aureus

[0088] For complement opsonization, pHrodo™ Green .S’. aureus BioParticles™ Conjugate (P35367, Thermo Fisher Scientific, Waltham, MA) were dissolved in 500 pL PBS, mixed with 500 pL of RPMI and opsonized with 250 pL human serum from a healthy donor for 1 hour at 37°C on a thermoshaker (400 rpm). After incubation, opsonized pHrodo™ Green .S', aureus BioParticles™ were pelleted by centrifugation and resuspended in 440 pL of RPMI.

[0089] Neutrophils were isolated from healthy subjects with Polymorphprep (Axis-Shield, Dundee, UK) density gradient as described previously (Lood et al., Nat. Med., 22:146-153). Isolated neutrophils (l x 106cells / mL) were incubated with or without different rhsB 1 compounds at 5 pM (B, C, D, and E) for 1 hour and stimulated with RNP-IC (10 pg / mL) for another 2 hours. At the end of incubation, neutrophils were pelleted by centrifugation and resuspended in 50 pL RPMIAvell at a concentration of 4 x 106cells / mL. Subsequently, 20 pL of complement-opsonized pHrodo™ Green .S'. aureus BioParticles were added to the neutrophils and incubated for another 5 minutes at 37°C on a plate thermoshaker (200 rpm). The plate was then placed on ice to stop further reaction and the relative amounts of pHrodo™ Green S. aureus BioParticles taken up by phagocytosis determined by flow cytometry (Beckman Coulter, Brea, CA). Cells were gated based on forward scatter (FSC) / side scatter (SSC) properties, and the mean fluorescence intensity (MFI)of the pHrodo was determined using FlowJo (Tree Star, Ashland, OR) (Lindner et al., BioTechniques, 2020, 68:245-250; Boero et al., Frontiers in Immunology, 2021, 12:635825). ix. Statistical Analysis

[0090] Paired / -test were used for samples assuming Gaussian distribution. For nonparametric analyses, Mann-Whitney U test and Wilcoxon signed-rank test were used. All analyses were performed in GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA), and considered statistically significant at p<0.05.2. Results i. Purity and Protease inhibitory activities of serpin Bl variants

[0091] All preparations of rhsBl and variants thereof were >95% pure as judged by Coomassie- stained SDS-PAGE gels (FIG. 1A, top, “Purified proteins”) and contained < 0.25 EU / mg protein. It has been previously demonstrated that wild-type rhsB 1 (code D) is a potent inhibitor of hNE, hPR3, and hCG but not NSP4, forming stable complexes with, and inhibiting the amidolytic activity of hNE, hPR3, and hCG at molar ratios of 2: 1 or lower (FIG. 1A, bottom three panels, and FIG. IB). The C344A variant (code C) was equally potent at inhibiting hCG but more effective at inhibiting hNE and hPR3 than the wild-type protein. In contrast, and as previously described, oxidized rhsB 1 (code A) was unable to inhibit hNE but retained hCG inhibitory activity although it was less potent than the wild-type protein (Pemberton, Protein expression and purification, 2020, 170:105595). In addition, it was found that it did not form stable complexes with hPR3 nor significantly inhibit its amidolytic activity. As predicted, reactive- site cleaved rhsBl (code B) and the T331R insertion-blocker variant (code F) had no significant inhibitory activity against any of the NSPs tested. These results are summarized in Table 1. ii. Serpin Bl inhibits IC-mediated NET formation

[0092] Although rhsBl has been previously demonstrated to block PMA, PAF, MIP-2 and LPS-mediated NET formation (Farley et al., J. Immunology, 2012, 189:4574-4581), its mechanism of action is unknown as is its ability to regulate pathophysiological relevant stimuli, including ICs which promote NET formation in autoimmune and infectious diseases. To investigate this, RNP-containing ICs, commonly seen in rheumatic disease and documented NET- inducing agonists, were used. For comparison, PMA was used to induce suicidal, NADPH-oxidase dependent NET formation, and the calcium ionophore A23187 to induce vital, NADPH- independent NET formation (Douda et al., PNAS, 2015, 112:2817-2822; Parker et al., Journal ofLeukocyte Biology, 2012, 92:841-849). It was found that wild-type rhsBl (code D), the C344A variant (code C), and oxidized rhsBl (code A) significantly and dose-dependently prevented IC- mediated NET formation (FIG. 2A-2B) and > 30-60% inhibition of DNA release and > 60-90% inhibition of DNA associated peroxidase activity were also consistently observed (over multiple experiments with independently isolated neutrophil preparations). Maximal inhibition was observed at concentrations > 250 nM. The data also demonstrated dose-dependent inhibition of PMA- and A23187-mediated NET formation by all three forms of the protein, in particular using the C334A variant (codes D, C and A, FIG. 2C-2F).Hi. Inhibition of NET formation by sBl requires an intact reactive center loop and the ability to undergo the S to R transition

[0093] Whether rhsB 1 inhibition of IC-mediated NET formation is dependent on having an intact reactive center loop (RCL) that could undergo the structural changes accompanying effective complex formation was next investigated. Cleaving rhsB 1 within the RCL induces the peptide sequence N-tcrminal to the reactive site to insert into 0-plcatcd sheet A and the protein to undergo a transition from a stressed (S) to relaxed (R) state which lacks any residual protease inhibitory activity. This form of rhsBl (code B) could not prevent NET formation indicating that an intact RCL is required (FIG. 2A-2F, FIG. 3A-3B). Whether the conformational change that accompanies protease inhibition is required for inhibition of NET formation was also investigated. For this experiment, the rhsBl T331R variant (code F) which possesses an intact RCL was used but, as shown in FIG. 1A, it cannot undergo the conformational change required for effective complex formation (FIG. 1A, no complex formed in lane F). This form of rhsBl also did not inhibit NET formation indicating that the ability to undergo this conformational change is required to block NET formation (FIG. 3A-3B). iv. Serpin Bl inhibition of IC-induced NET formation is dependent on its chymotrypsin- like inhibitory activity

[0094] In an attempt to define what, if any, protease(s) inhibited by rhsB 1 are involved in NET formation induced by these agents, two variants of rhsBl with differing protease inhibitory activities were tested, oxidized rhsB 1 (code A) which does not inhibit hNE or hPR3 but retains hCG inhibitory activity and an engineered rhsB 1 which specifically inhibits hPR3 (and hNE) but does not inhibit hCG (code G). It was found that oxidized rhsBl still blocked IC-induced NET formation whereas the hPR3-specific inhibitor had no effect on NET formation induced by any ofthe agents tested. This suggests that rhsBl blocks IC-mediated NET formation in a hNE- and hPR3-independent manner and that inhibition of a chymotrypsin-like enzyme, perhaps hCG, is involved.

[0095] To further examine the nature of this inhibition, AAT (code E), the small molecule elastase inhibitor sivelestat (CAS # 201677-61-4), which is cell permeable and can inhibit NET formation in vitro in a hNE-dependent manner (Okeke et al., Biomaterials, 2020, 238:119836), a non-cell permeable specific competitive, non-peptide inhibitor of hCG (Cathepsin G inhibitor I; CAS # 429676-93-7; Sigma-Aldrich cat # 219372) (Greco et al., J. Am. Chem. Soc., 2002, 124:3810-3811), and the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) were tested. While AAT can inhibit hCG, it had no significant effect on NET formation (FIG. 3A-3B). In contrast, sivelestat had small (10-20%) but significant inhibitory effects on DNA release and peroxidase activity. Cathepsin G inhibitor I blocked both DNA release and peroxidase activity by up to 50%, a level comparable to that routinely achieved with rhsBl (code A, C or D) although 4- fold higher levels of the inhibitor were used (FIG. 3A-3B, “CatG inh”). DPI almost completely blocked IC- and PMA-mediated DNA release and peroxidase activity consistent with earlier reports that these are ROS-dependent events. In contrast, DPI had little effect on A23187- mediated peroxidase release (FIG. 2F) but inhibited DNA release by up to 50% (FIG. 2E), consistent with earlier reports that this is a ROS-independent event. v. Treatment with rhsBl C344A amplifies neutrophil adhesion molecules and ROS formation

[0096] Given the capacity of rhsBl and select variants to block NET formation induced by three distinct stimuli, whether they altered the levels of other specific markers of neutrophil activation or impacted ROS formation was then investigated. It was found that wild-type rhsB 1 (code D) (and AAT (code E)) had no significant effect on the neutrophil surface levels of activated CDllb, CD66b, or CD63 (FIG. 4B-4D). It did slightly elevate ROS production, this result however was not statistically significant (FIG. 4A). In contrast, the C344A variant (code C) was the only agent tested that significantly elevated all three neutrophil markers by about 200% or more (FIG. 4B-4D) and increased intracellular ROS production 2.5-fold (FIG. 4A). Sivelestat had no effect on CDl lb, CD66b, CD63 or ROS production (FIG. 4A-4D). As expected, DPI completely abolished ROS production (FIG. 4A). Thus, whereas NET formation was prevented by the C344A variant, other neutrophil effector functions were enhanced, suggesting a skewing ofthe neutrophil activation response upon treatment with the C344A variant but not wild-type rhsB 1 or other elastase inhibitors. vi. Serpin Bl enhances microbial phagocytosis but not Immune complex clearance

[0097] Because the rhsBl C344A variant specifically boosted neutrophil surface levels of molecules involved in cellular adhesion, migration, and phagocytosis and prior studies suggest that serpin Bl, like AAT, may suppress bacterial proliferation (Woods et al., Pediatric Pulmonology, 2005, 39:141-149; Cantin et al., Am. J. Respir. Crit. Care Med., 1999, 160:1130-1135), whether rhsB 1 (code D) or selected variants (codes B and C) could impact neutrophil phagocytosis of either ICs or bacteria was investigated. It was found that none of the reagents tested were able to significantly boost phagocytosis of ICs, but several were able to boost phagocytosis of complement-opsonized pHrodo™ Green-labeled .S', aureus BioParticles (FIG. 5A-5B). AAT, and rhsBl WT (code D) and the C344A variant (code C) all significantly increased uptake of these particles by 20-30%. In contrast, the RCL-cleaved form of the protein (code B) had no effect.3. Conclusion

[0098] This study was set out to follow-up on the 10-year-old observations that rhsBl can inhibit NET formation (Farley et al., Journal of Immunology, 2012, 189:4574-4581) and extend those observations to include NET-formation induced by RNP-IgG ICs - the main driver of NET formation in many rheumatic diseases. It was also sought to determine a mechanism of action by using rhsBl variants with altered protease inhibitory activity and specificities. For the related serpin AAT, many of its immunomodulatory activities are unrelated to its ability to bind and inhibit proteases, but rather depend on direct binding and sequestration of other ligands that drive inflammation within hydrophobic pockets present on the surface of the molecule (e.g. LTB4) (O’Brien et al., hit. J. Mol. Sci., 2022, 23(5):2441; O’Dwyer et al., Journal of Immunology, 2015, 195:3628-3641). Furthermore, AAT and sBl both inhibit all of the major NSPs, yet AAT does not inhibit NET formation whereas sBl does, thus it is not obvious that protease inhibition would necessarily be a part of the mechanism by which rhsBl blocks NET formation. Nevertheless, several lines of evidence point to the involvement of an NSP, and in particular, hCG, in mediating IC-driven NET formation in neutrophils. First, non-protease inhibitory valiants of rhsB 1 do not block NET formation. Second, rhsBl variants that only inhibit hNE and PR3 also do not block NET formation whereas rhsBl variants that specifically inhibit chymotrypsin-like proteases via F343 do block NET formation. Third, the concentrations and dose-dependency of rhsB 1 requiredto inhibit NET formation are similar to the levels of intracellular NSPs available for release during neutrophil degranulation and much higher than typically required for activation of ligand-mediated receptor-mediated signaling pathways. Fourth, pre-incubation with a specific cell impermeable hCG inhibitor also blocks NET formation. Finally, rhsBl must be present prior to the addition of the NET inducing agent. Adding it at the time of stimulation or later has no effect (Farley et al., Journal of Immunology, 2012, 189:4574-4581) suggesting that a molecule released during early neutrophil activation may contribute to NET formation.

[0099] Additional evidence supporting this hypothesis comes from observations made using variants of another major neutrophil protease inhibitor - secretory leukocyte protease inhibitor (SLPI). SLPI is a low molecular weight protein protease inhibitor (11.7 kDa) that also inhibits hCG and hNE, and blocks NET formation. Its mechanism of action has been partly attributed to its anti-hNE activity (Sallenave et al., Journal of Leukocyte Biology, 1997, 61:695-702; Zabieglo et al., Journal of Leukocyte Biology, 2015, 98:99-106). However, like rhsBl, SLPI variants lacking hNE inhibitory activity still inhibit NET formation to some extent and retain some inhibitory potential against chymotrypsin (hCG) or trypsin-like proteases. Although the definitive protease responsible for inducing NET formation was never identified in this study, the data support a role for a protease other than hNE, perhaps hCG (Zabieglo et al., Journal of Leukocyte Biology, 2015, 98:99-106). Other studies in mouse models of deep-vein thrombosis also show that elastase is not required for NET formation and subsequent production of pathological venous thrombi (Martinod et al., J. Thromb. Haemost., 2016, 14(3):551-558). More recently, an indirect role for hCG in activating platelet-mediated NET formation and thrombosis has emerged (Carnevale et al., Arterioscler. Thromb. Vase. Biol., 2023, 43(10):e396-e403). Collectively, these findings support the involvement of NSPs other than elastase in inducing NET formation and provide evidence that hCG may play a role early on in the process of NET formation. Multiple pro-inflammatory mechanisms of action have been ascribed to hCG during inflammatory and autoimmune diseases (Gao et al., Archives of Rheumatology, 2018, 33:498-504; Pham et al., Nat. Rev. Immunol., 2006, 6:541-550) and while previous studies have revealed a clear role for intracellular sBl in preventing hPR3-mediated apoptosis and hCG-mediated necrosis, to the inventors’ knowledge, this is the first study to suggest that hCG may play a role in directly triggering NET formation and that exogenous sB 1 may prevent that. Follow-up studies evaluatingthe direct effect of hCG on NET formation should help further elucidate how it triggers NET formation.

[0100] Based on these observations, it is proposed that a chymotrypsin-like protease, most likely hCG, released during neutrophil activation by ICs (and other stimulants) acts at the cell surface to induce NET formation (FIG. 6). How it induces NET formation is currently unknown, however it must be dependent on its proteolytic activity as both rhsB 1 and the small molecule hCG inhibitor prevent NET formation. AAT also inhibits hCG yet does not inhibit NET formation induced by ICs or other activating agents. Several reasons for this may be: 1) the larger size of glycosylated AAT (54 kDa) vs non-glycosylated rhsBl (42 kDa) sterically hindering access to hCG bound to the surface of neutrophils (Owen et al., Journal of Cell Biology, 1995, 131:775- 789); 2) differences in the rate or stability of inhibition of hCG by the respective inhibitors - rhsB 1 inhibits hCG 5-fold faster than AAT and this reaction is not markedly affected by oxidation whereas the AAT:hCG reaction rate is reduced almost 1,000-fold (Cooley et al., Biochemistry, 2001, 40:15762-15770; Beatty et al., Journal of Biological Chemistry, 1980, 255:3931-3934), or 3) interactions of other domains on rhsBl that are not present in AAT with cell surface receptors preventing triggering of programmed cell death pathways.

[0101] Several hCG targets on the neutrophil surface or released by neutrophils that are, or have the potential to be, involved in activation of NET formation are the PARs (protease activated receptors), integrins, G-protein coupled receptors, and chemokines / cytokines. For example, PAR- 2 cleavage and activation by a class of bacterial-derived cysteine proteases called gingipains directly results in NET formation (Bryzek et al., PLoS Pathogens, 2019, 15:el007773). In contrast, PAR-2 is also cleaved by the NSPs with varying and biased effects on PAR-2-mediated signaling (Pham, hit. J. Biochem. Cell Biol., 2008, 40:1317-1333; Ramachandran et al., Journal of Biological Chemistry, 2008, 286:24638-24648). Ramachandran et al. found that all three NSPs disarmed PAR-2 by cleaving and releasing the N-terminal tethered ligand (TL) sequence thereby preventing signaling by other proteases. However, only elastase induced a TL- independent PAR- 2 activation of MAP kinase and selective activation of the p44 / 42 MAPK pathway demonstrating that NSPs can signal independently to effect changes intracellularly. hCG has been shown to modulate neutrophil spreading and the clustering of integrins leading to optimal neutrophil effector functions (Raptis et al., Immunity, 2005, 22:679-691). It is also a potent chemoattractant and exerts it affects by binding to formyl peptide receptor (FPR) inducing a Ca2+flux and translocationof PKC^ (Sun et al., Journal of Immunology, 2004, 173:428-436). hCG can also modulate the release and activity of chemokines and cytokines (Pham, Int. J. Biochem. Cell Biol., 2008, 40:1317-1333). Additional experiments are required to help understand which, if any, of these pathways arc affected by rhsBl during NET formation.

[0102] Oxidation did not markedly affect the ability of wild-type rhsBl to block NET formation or induce significant changes in levels of neutrophil activation markers or ROS production. In contrast, an unexpected finding was that the oxidation-resistant C344A variant gave very different results. It significantly boosted levels of all three markers and ROS production while effectively suppressing NET formation. It is not currently understood how this particular' variant boosts neutrophil surface levels of these markers but the increase in activated CD1 lb may help explain the observed increase in ROS production. NSPs and in particular, hNE, are capable of degrading cell surface receptors required for efficient assembly of the NADPH oxidase (NoX) and phagocytosis including Fey receptors (FcyRIII), CR1 (but not CDl lb / CD18 [CR3, Mac-1, UMP2]), and opsonins (iC3b) and opsonin receptors (Tosi ct al., Journal of Clinical Investigation, 1990, 86:300-308; Tosi et al., Journal of Clinical Investigation, 1992, 90:462-470; Berger et al., Journal of Clinical Investigation, 1989, 84:1302-1313). Previous studies have shown that CD1 lb / CD18 works in concert with FcyRIII to generate the neutrophil respiratory burst and has a requirement for tyrosine phosphorylation on the IT AM (immunoreceptor tyrosine-based activation motif) domain of FcyRIIA and subsequent signaling (Zhou et al., Journal of Cell Biology, 1994, 125:1407-1416; Zhou et al, Journal of Biological Cemistry, 1995, 270:13553-13560). Thus, hNE shedding of FcyRIII (or FcyRIIA) would render it unable to drive assembly of the NoX complex while inhibition of hNE by effective elastase inhibitors including the C344A variant should prevent that. However, silvelestat and AAT also had no effect on ROS production most likely because they did not boost CD1 lb activation and optimal ROS production necessarily requires both CD1 lb and FcRIII.

[0103] Other proteases also contribute to the shedding of neutrophil cell surface receptors. The main neutrophil matrix metalloprotease responsible for shedding FcyRIIIB is ADAM 17 (a disintegrin and metalloprotease). ADAM 17 is induced by multiple stimulants including fMLP, PMA and FcR / CR-dependent phagocytosis and its activation requires caspase 8 and production of ROS by mitochondria. Similarly, RNP-IC activation of neutrophil TLR 7 / 8 induces furin- mediated shedding of FcyRIIA reducing phagocytic activity of ICs while increasing theirpropensity to undergo NET formation (Lood et al., J. Exp. Med., 2017, 214:2103-2119). It is not yet known the effect of rhsBl on ADAM17 or furin activity but in relation to TLR 7 / 8 signaling, CD 11b activation has been shown to reduce inflammation and autoimmunity in SLE by suppressing TLR and IFN-I signaling via negative regulation of the NF-KB pathway and increased degradation of MyD88 (Faridi et al., Journal of Clinical Investigation, 2017, 127:1271-1283; Han et al., Nature Immunology, 2017, 11:734-742).

[0104] Given the role of CDl lb / CD18 in phagocytosis, it is hypothesized that adding rhsBl to IC-treated neutrophils would not only prevent NET formation but may boost their uptake of RNP-ICs and / or opsonized bacterial particles. While improved uptake of RNP-ICs were not observed, increased phagocytosis of complement-opsonized pHrodo-labelled 5. aureus bioparticles by neutrophils treated with both wild-type and C344A rhsB 1 and AAT but not RCL- cleaved rhsBl, was observed. As mentioned earlier, hNE can degrade both CR1 and opsonins but not CDl lb / CD18, thus effective inhibition of hNE by all three proteins may boost phagocytosis via CR1. However, the marked increase in CDl lb-drivcn by the C344A variant alone did not result in higher phagocytosis than that achieved with wild-type rhsBl thus CD1 lb alone does not appear to have a role to play in enhancing phagocytosis. Aside from boosting ROS production, further studies evaluating the role of elevated CDl lb / CD18 (and CD63 and CD66b) on other neutrophil effector functions such as adhesion and migration are warranted.

[0105] In conclusion, a mechanism of action by which exogenously added rhsBl prevents NET formation induced by multiple stimuli including RNP-IC has been uncovered in this study. The mechanism is dependent upon inhibition of a chymotrypsin-like protease, and studies with specific inhibitors indicate that hCG is the protease responsible. This study also unexpectedly uncovered a potential mechanism whereby the C344A variant boosted ROS production and clearance of opsonized particles while simultaneously suppressing NET formation.

[0106] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the protein constructs, methods, and / or component features, steps, elements, or other aspects thereof can be used in various combinations.

[0107] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any clcmcnt(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects of the disclosure, is / are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0108] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

We Claim:

1. A method of treating a disease or disorder associated with neutrophil extracellular trap (NET) formation, said method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G.

2. The method of claim 1, wherein the molecule capable of inhibiting cathepsin G is selected from a peptide, a polypeptide, or a non-peptide small molecule, optionally wherein the molecule capable of inhibiting cathepsin G is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non- structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, or an elastin-like peptide, or is PEGylated.

3. The method of claim 1 or 2, wherein the molecule capable of inhibiting cathepsin G comprises or is cathepsin G inhibitor I having a CAS registry number of CAS 429676-93-7.

4. The method of claim 1 or 2, wherein the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide, an oxidized serpin B 1 polypeptide, or a serpin B 1 variant polypeptide, optionally wherein the serpin Bl polypeptide, oxidized serpin Bl polypeptide, or serpin Bl variant polypeptide is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non- structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, or an elastin-like peptide, or is PEGylated.

5. The method of claim 4, wherein the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

6. The method of claim 5, wherein the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

7. The method of claim 4, wherein the serpin B 1 variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344.

8. The method of claim 7, wherein the amino acid substitution is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1.

9. The method of claim 8, wherein the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

10. The method of any one of claims 1-9, wherein the disease or disorder is a relapsingremitting disease or disorder associated with NET formation, and the subject is in remission at the time of treatment.

11. The method of claim 10, wherein administration of the molecule capable of inhibiting cathepsin G prolongs remission or prevents or delays onset of a relapse of the disease or disorder.

12. The method of any one of claims 1-11, wherein the disease or disorder is an immune complex driven autoimmune disease.

13. The method of claim 12, wherein the disease or disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

14. The method of any one of claims 1-9, wherein the disease or disorder involves NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis.

15. A method of preventing or reducing recurrence and / or severity of a bacterial infection in a subject suffering from episodic recurrent bouts of inflammation associated with neutrophil extracellular trap (NET) formation, said method comprising administering to the subject a therapeutically effective amount of a serpin Bl variant polypeptide, wherein the serpin Bl variant polypeptide is oxidation-resistant, optionally wherein the serpin B 1 variant polypeptide is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non-structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine- serine polymer, or an elastin-like peptide, or is PEGylated, and optionally wherein the subject is immunocompromised.

16. The method of claim 15, wherein administration of the serpin Bl variant polypeptide increases phagocytosis and boosts intracellular reactive oxygen species (ROS) production in the subject.

17. The method of claim 15 or 16, wherein the serpin Bl variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344.

18. The method of claim 17, wherein the amino acid substitution is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1.

19. The method of claim 18, wherein the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

20. The method of any one of claims 15-19, wherein the episodic recurrent bouts of inflammation are caused by a relapsing-remitting disease or disorder associated with NET formation, and the subject is in remission at the time of treatment.

21. The method of claim 20, wherein administration of the serpin Bl variant polypeptide prolongs remission or prevents or delays onset of a relapse of the disease or disorder.

22. The method of claim 20 or 21, wherein the relapsing-remitting disease or disorder associated with NET formation is multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), antineutrophilic cytoplasmic antibody (ANCA) associated vasculitis (AAV), small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.

23. The method of any one of claims 15-19, wherein the subject is diagnosed with a disease or disorder involving NET formation, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis.

24. A method of preventing or delaying onset of a relapse of a disease or disorder, wherein the relapse is mediated by neutrophil extracellular trap (NET) formation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a molecule capable of inhibiting cathepsin G.

25. The method of claim 24, wherein the molecule capable of inhibiting cathepsin G is selected from a peptide, a polypeptide, or a non-peptide small molecule, optionally wherein the molecule capable of inhibiting cathepsin G is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non- structured, inert polypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, or an elastin-like peptide, or is PEGylated.

26. The method of claim 24 or 25, wherein the molecule capable of inhibiting cathepsin G comprises or is cathepsin G inhibitor I having a CAS registry number of CAS 429676-93-7.

27. The method of claim 24 or 25, wherein the molecule capable of inhibiting cathepsin G comprises or is a serpin B 1 polypeptide, an oxidized serpin B 1 polypeptide, or a serpin B 1 variant polypeptide, optionally wherein the serpin Bl polypeptide, oxidized serpin Bl polypeptide, or serpin Bl variant polypeptide is fused to a constant fragment (Fc) domain of a human immunoglobulin G, a human serum albumin, a human transferrin, or a non- structured, inertpolypeptide, such as XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, or an elastin-like peptide, or is PEGylated.

28. The method of claim 27, wherein the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

29. The method of claim 28, wherein the serpin Bl polypeptide or oxidized serpin Bl polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

30. The method of claim 27, wherein the serpin Bl variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at residue 344.

31. The method of claim 30, wherein the amino acid substitution is C344A, C344G, C344V, or C344S as compared to the sequence of SEQ ID NO: 1.

32. The method of claim 31, wherein the serpin Bl variant polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

33. The method of any one of claims 24-32, wherein the disease or disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, antineutrophilic cytoplasmic antibody associated vasculitis, small vessel vasculitis, IgA nephropathy, sepsis, type-1 diabetes, gout, heparin-induced thrombosis, immune complex induced thromboses, myasthenia gravis, or psoriasis.