Novel therapeutic polypeptides

Abstract

This disclosure provides alpha-1 antitrypsin (AAT) polypeptides as defined herein. Also provided herein are pharmaceutical compositions comprising the polypeptide, nucleic acids encoding the polypeptide, vectors comprising the nucleic acid, cells, methods for producing the AAT polypeptide, and methods for using the AAT polypeptide in therapy.

Description

[Technical Field]

[0001] This disclosure relates to alpha-1 antitrypsin (AAT) polypeptides. This disclosure also relates to pharmaceutical compositions comprising the polypeptide, nucleic acids encoding the polypeptide, vectors comprising the nucleic acid, cells, methods for producing the AAT polypeptide, and methods for using the AAT polypeptide in therapies. [Background technology]

[0002] The lungs are constantly exposed to various infectious and environmental substances, which can potentially damage them. This triggers an immune response associated with inflammation. While this inflammatory response is a defense against these threats, if excessive, it can contribute to lung damage and clinical disease. Some important examples include chronic obstructive pulmonary disease (COPD) / emphysema, cystic fibrosis (CF), pulmonary fibrosis, air pollution (a cause of approximately 9 million deaths annually), and various infections such as bacterial (e.g., Haemophilus influenzae) and viral (e.g., influenza).

[0003] While the inflammatory / immune response in the lungs is complex, a key pathway is the production of proteases such as neutrophil elastase (NE). Overproduction of proteases and / or deficiencies in inhibitors lead to protease imbalance and lung damage. This protease imbalance is also associated with inflammatory conditions in other parts of the body, such as sepsis, and with certain cancers (e.g., lung cancer).

[0004] A key mechanism of protease expression in the lungs is the formation of extracellular traps. These traps are expressed by phagocytic cells such as neutrophils (NETs) and other cells such as macrophages (METs), and are antimicrobial. Traps consist of extracellular chromatin (DNA) and co-expressed mediators such as NE. NETs play a crucial role in activating the inflammatory response to eliminate such infections. However, components of the traps, such as proteases, may be damaging host tissues. Recent studies suggest that NETs may cause significant tissue damage in other inflammatory diseases.

[0005] One of the important antiproteases produced in the body is serpine alpha-1 antitrypsin (AAT). AAT inactivates serine proteases such as NE and trypsin (and also downregulates other proteases such as metalloproteinase 9).

[0006] While AAT acts on proteases, it also possesses a broader range of anti-inflammatory effects that are not fully understood or adequately classified. These effects include the reduction of cytokines such as interleukin-8 (IL-8) and tumor necrosis factor, inflammatory intracellular signaling pathways, and apoptosis. Due to its effects on NE and IL-8 as well as signaling pathways, AAT has a specific effect of downregulating neutrophil-driven inflammation. In contrast to other cellular pathways, such as those driven by eosinophils, there are few biological therapies that specifically target the effects of neutrophils.

[0007] AAT also has a direct antiviral effect by interfering with host proteases that viruses such as influenza and coronaviruses use to infect the respiratory epithelium. Therefore, AAT can be considered not only an anti-inflammatory agent but also an antiviral agent for some infections.

[0008] AAT is one of the most common proteins in human serum / plasma, and blood-derived AAT has been used for many years in the United States (and other countries) as chronic replacement therapy for patients with clinical conditions such as birth defects and emphysema. In this context, AAT has been shown to be very safe and well-tolerated. AAT is approved for patient use by the Therapeutic Goods Administration. AAT is not used as an anti-inflammatory agent.

[0009] While AAT (arteriovenous adenomatous thromboembolism) holds great potential as a therapy for a variety of common medical conditions, it is actually rarely used. Significant problems limiting its use include its severely limited availability due to its origin from blood donors, and its extremely high cost (approximately $100,000 per year in Australia). AAT is also highly heterogeneous and unstable.

[0010] Not all proteins fold to the most thermodynamically stable conformation; instead, they pause folding upon reaching certain intermediates. AATs fold to a metastable native conformation, using this metastable state to drive their function, which involves an irreversible but thermodynamically favorable structural transition (known as the S-to-R transition) to a more stable cleavage state. Due to the inherent metastable state of the native functional state, AATs are susceptible to misfolding and aggregation under various conditions, including heat and the presence of mutations. Therefore, improving the stability (both thermodynamic and kinetic) of the metastable native state can prevent misfolding and aggregation without affecting its inhibitory function. Improving AAT protein stability is important for the development of novel AAT therapies, as protein stability is a critical factor for AAT therapy, especially when using administration routes requiring aerosol delivery. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] AATs represent a wide range of therapies that may be beneficial for a vast number of inflammatory lung diseases (e.g., COPD, CF, pulmonary fibrosis, air pollution, and infections such as influenza and Haemophilus influenzae) and extrapulmonary conditions, but are currently unavailable (in addition to their established efficacy as supplemental treatments in patients with birth defects). It is desirable to develop recombinant forms of AATs that can be used for one or more of these diseases. Furthermore, it is desirable to understand the effect of various mutations on the metastability of AATs. Moreover, it is desirable to provide modified AATs with improved stability and folding properties that are useful for therapy, relatively homogeneous, and / or can be commercially manufactured in reasonable quantities at competitive cost. [Means for solving the problem]

[0012] In one embodiment, an alpha-1 antitrypsin (AAT) polypeptide is provided, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, wherein the amino acid sequence differs from SEQ ID NO: 2 in that it contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

[0013] In some embodiments, the amino acid sequence is the following group of amino acid substitutions from SEQ ID NO: S276K, T278E, T323E, D325N and K327E, L275F, I324V and M358I, and G99A, T144W, and Y171A It has one, two, or all of them.

[0014] In some embodiments, the amino acid sequence has 5 or more, or 6 or more, or 8 or more amino acid substitutions selected independently from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, from SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence includes each of the following amino acid substitutions from SEQ ID NO: 2: G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

[0016] In some embodiments, the amino acid sequence includes one or more additional amino acid substitutions from SEQ ID NO: 2 selected independently from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R.

[0017] In some embodiments, the amino acid sequence includes 2 or more, or 3 or more, or 4 or more, or 5 or more, or each of the following additional amino acid substitutions from SEQ ID NO: 2: F35L, T43A, T52A, A54G, S365A, and K371R.

[0018] In some embodiments, the amino acid sequence has at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO: 2.

[0019] In some embodiments, the amino acid sequence has 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer amino acid modifications from SEQ ID NO: 2.

[0020] In some embodiments, the amino acid sequence is Comprising 3 or more amino acid substitutions from SEQ ID NO: 2 independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, and Optionally, comprising 1 or more additional amino acid substitutions from SEQ ID NO: 2 independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R In addition, having 10 or fewer, 5 or fewer amino acid modifications from SEQ ID NO: 2, or having no amino acid modifications.

[0021] In some embodiments, the polypeptide comprises additional N-terminal and / or C-terminal amino acid sequences.

[0022] In some embodiments, prior to the amino acid sequence, the AAT polypeptide comprises the N-terminal sequence MENLYFQGAAS (SEQ ID NO: 7) or MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHH (SEQ ID NO: 8).

[0023] In some embodiments, the AAT polypeptide has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0024] In some embodiments, the AAT polypeptide has improved thermal stability compared to wild-type human AAT.

[0025] In some embodiments, the AAT polypeptide has an improved refolding yield compared to wild-type AAT.

[0026] In some embodiments, the AAT polypeptide comprises a modification selected from the group consisting of glycosylation, PEGylation, prenylation, acylation, biotinylation, phosphorylation, and conjugation to a lipid moiety.

[0027] In another aspect, a nucleic acid molecule encoding an AAT polypeptide as defined herein is provided.

[0028] In another embodiment, a vector is provided that includes a nucleic acid sequence encoding an AAT polypeptide as defined herein.

[0029] In some embodiments, the nucleic acid sequence is linked to an expression regulatory sequence suitable for expression in the host cell.

[0030] In another embodiment, cells containing nucleic acids as defined herein are provided.

[0031] In some embodiments, the cells include a vector as defined herein.

[0032] In some embodiments, the cells are mammalian cells or bacterial cells.

[0033] In some embodiments, the cells are DH5α, BL21(DE3)pLysS, SG13009, or Rosetta Blue DE3 cells.

[0034] In another embodiment, a method for producing an AAT polypeptide as defined herein, a. Transfecting host cells with a nucleic acid sequence encoding an AAT polypeptide as defined herein, b. Transfected host cells are cultured in cell culture medium and express the AAT polypeptide. c. Recovering AAT polypeptide from cell culture medium and A method including this is provided.

[0035] In another embodiment, a pharmaceutical composition is provided comprising an AAT polypeptide as defined herein and a pharmaceutically acceptable excipient.

[0036] In some embodiments, the pharmaceutical composition retains at least 80%, at least 90%, or at least 95% of its initial neutrophil elastase inhibitory activity after storage at 4°C for 3 months or 6 months.

[0037] In another embodiment, an AAT polypeptide as defined herein, or a pharmaceutical composition as defined herein, for use as a pharmacopoeia is provided.

[0038] In some embodiments, the AAT polypeptide or pharmaceutical composition is intended for use in the prevention or treatment of AAT deficiency or diseases or disorders associated with AAT deficiency.

[0039] In some embodiments, the AAT polypeptide or pharmaceutical composition is intended for use in the treatment or prevention of inflammatory diseases, respiratory diseases, cancer and / or viral infections.

[0040] In some embodiments, inflammatory diseases, respiratory diseases and / or viral infections are selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory diseases, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, respiratory diseases associated with air pollution, cancers associated with protease activity (e.g., lung cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

[0041] In another embodiment, a method is provided for preventing or treating AAT deficiency, or a disease or disorder associated with AAT deficiency, in a subject, comprising administering to the subject an effective amount of an AAT polypeptide as defined herein, or a pharmaceutical composition as defined herein.

[0042] In some embodiments, the method is for treating or preventing inflammatory diseases, respiratory diseases, cancer, and / or viral infections.

[0043] In some embodiments, inflammatory diseases, respiratory diseases and / or viral infections are selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory diseases, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, respiratory diseases associated with air pollution, cancers associated with protease activity (e.g., lung cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

[0044] In another embodiment, the use of an AAT polypeptide, as defined herein, for the manufacture of a drug for preventing or treating AAT deficiency or a disease or disorder associated with AAT deficiency is provided.

[0045] In some embodiments, the drug is used to treat or prevent inflammatory diseases, respiratory diseases, cancer, and / or viral infections.

[0046] In some embodiments, inflammatory diseases, respiratory diseases and / or viral infections are selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory diseases, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, respiratory diseases associated with air pollution, cancers associated with protease activity (e.g., lung cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

[0047] In some embodiments, the method, the use, or the AAT polypeptide or pharmaceutical composition for such use achieves one or more of the following: reduction of cytokine levels in the target, reduction of extracellular trap formation in the target organ, reduction of protease activity, and reduction of leukocyte infiltration in the target organ.

[0048] In some embodiments, the disease or disorder is an inflammatory disease or disorder, and is a neutrophil-related inflammatory disease or disorder.

[0049] In some embodiments, the method, use, or use of the AAT polypeptide or pharmaceutical composition comprises administering the AAT polypeptide or pharmaceutical composition in combination with a further therapeutic agent. [Brief explanation of the drawing]

[0050] [Figure 1] This figure shows sequence information related to this disclosure. Sequence ID 1 shows the amino acid sequence for wild-type human AAT (wtAAT). Sequence ID 2 shows the amino acid sequence for the section of the wtAAT sequence on which the AAT polypeptide of this disclosure is based. Sequence ID 3 shows the amino acid sequence for sAAT, an exemplary AAT polypeptide. The difference from the corresponding sequence of wtAAT is shown in bold. This sequence has an E coli N-terminal tag. Sequence ID 4 shows the amino acid sequence for sAAT, an exemplary AAT polypeptide. The difference from the corresponding sequence of wtAAT is shown in bold. This sequence has a CHO N-terminal tag. Sequence ID 5 shows the amino acid sequence for uAAT, a further exemplary AAT polypeptide. The difference from wtAAT is shown in bold. This sequence has an E coli N-terminal tag. Sequence ID 6 shows the amino acid sequence for uAAT, a further exemplary AAT polypeptide. The difference from the corresponding sequence of wtAAT is shown in bold. This sequence has a CHO N-terminal tag. SEQ ID NO: 7 shows the amino acid sequences of the N-terminal E coli sequences used in two exemplary AAT polypeptides, sAAT and uAAT, of this disclosure. SEQ ID NO: 8 shows the amino acid sequences of the N-terminal sequence of wild-type human AAT produced in a CHO expression system, as well as the N-terminal sequences used in two exemplary AAT polypeptides, sAAT and uAAT, of this disclosure. SEQ ID NO: 9 shows the amino acid sequences of additional N-terminal sequences used in embodiments of this disclosure. [Figure 2]Figure 2A shows the results of in vitro and in vivo models using mist-type wtAAT, demonstrating its potent anti-inflammatory effect. Figure 2A shows the effect of wtAAT administration on white blood cell count (WCC), and Figure 2B shows the effect of wtAAT administration on lung weight. [Figure 3A] This figure shows a front view of regions grafted onto wtAAT (PDB ID: 3NE4). Each region grafted onto wtAAT contributes to biophysical properties such as improved thermal stability and / or kinetic stability, and / or reduced aggregation tendency, and / or improved colloidal properties, through various favorable interactions. Mutant residues are shown as rod-shaped material (wtAAT = white, mutant residues = black). Top left: F51: Improved packing for β-sheet A; Top right: Citrate bond: Stabilizes the native state; Bottom left: Breach: Increased salt bridges for improved stability; Bottom right: Helix-F: Improved packing and added rigidity. [Figure 3B] This figure shows the back view of regions grafted onto wtAAT (PDB ID: 3NE4). Each region grafted onto wtAAT is assumed to contribute to ideal biophysical properties through various favorable interactions. Mutant residues are shown as rod-shaped material (wtAAT = white, mutant residues = black). Top left: β-sheet C stapling: further salt bridges to stabilize the sheet; Top right: Helix-H: improved packing, additional salt bridges for further stabilization; Bottom left: T59: favorable polar and nonpolar interactions; Bottom right: B / C barrel: improved hydrophobic packing. [Figure 4]The results of biophysical analysis of sAAT, the AAT polypeptide produced in the CHO expression system according to this disclosure, are shown. A. Spectroscopic scans before and after thermal denaturation show structural changes after melting. B. Thermal denaturation curves of each triple repeat that produce a midpoint (Tm) of melting at 73.4°C. C. Inhibitory activity against HNE indicates that the sAAT graft is active against HNE. D. The yield of monomeric protein is higher than that of wtAAT and individual thermally stable grafts, respectively. E. Exogenous fluorescence indicates that the folding intermediate is present at a higher denaturation concentration for the sAAT graft. [Figure 5] This figure shows the effects of three different forms of AAT polypeptide produced in a CHO expression system, referred to as uAAT, sAAT, and rAAT, and their ability to reduce NE activity in IAV-infected neutrophils. All three forms of AAT resulted in a similar and significant reduction in NE activity. [Figure 6] This figure shows a graph illustrating the change in neutrophil elastase activity upon administration of the AAT polypeptide, referred to as sAAT, produced in an E. coli expression system according to this disclosure. The standard curve shows that neutrophil elastase activity is reduced by sAAT (n=6, *p<0.05). [Figure 7] The chart shows the results of an in-vitro NET model with NTHi infection, illustrating that administration of the AAT polypeptide (sAAT) produced in the E. coli expression system, or administration of eluted wild-type AAT (referred to as eAAT), significantly reduces neutrophil elastase activity. [Figure 8] This figure shows a chart illustrating the changes in neutrophil elastate activity in human blood neutrophils after incubation with carbon black nanoparticles followed by administration of the AAT polypeptide (sAAT) produced in an E. coli expression system according to this disclosure. [Figure 9]This figure shows a chart illustrating the changes in neutrophil elastate activity in human blood neutrophils after incubation with silica nanoparticles followed by administration of the AAT polypeptide (sAAT) produced in the E. coli expression system according to this disclosure. [Figure 10] This figure shows a chart illustrating the changes in neutrophil elastate activity in human blood neutrophils after incubation with tobacco smoke extract (CSE) followed by administration of the AAT polypeptide (sAAT) produced in the E. coli expression system according to this disclosure. [Figure 11] This figure shows a plaque assay image demonstrating the ability of the recombinant AAT polypeptide (sAAT) produced in the E. coli expression system according to this disclosure to reduce / prevent influenza A virus infection in an epithelial model. The purple / dark assay indicates no infection. [Figure 12] This figure shows a chart illustrating the results of assays demonstrating the ability of AAT polypeptides produced in the E. coli expression system according to this disclosure (sAAT) and eluted wild-type AAT (referred to as eAAT) to inhibit neutrophil elastase activity after neutrophil infection with influenza A, after storage of AAT polypeptides in a refrigerator for 2 weeks or 5 months. [Figure 13] This figure shows the results of assays demonstrating the dose-response relationship between the three AAT polypeptides (uAAT, sAAT, and rAAT) produced in the CHO expression system according to this disclosure, and graphs showing their ability to inhibit neutrophil elastase activity. [Figure 14] This figure shows a chart illustrating the assay results demonstrating the ability of the AAT polypeptide (sAAT) produced in the CHO expression system according to this disclosure to inhibit neutrophil elastase activity in influenza A-treated neutrophils, both fresh and after 1 week of storage at ambient temperature. [Figure 15]A) A chart showing the results of an assay demonstrating the ability of the AAT polypeptide (sAAT) produced in a CHO expression system to reduce neutrophil elastase activity when treated with fresh sAAT and sAAT stored at ambient temperature for one week at various dosages. B) A graph showing the results of an assay demonstrating the dose-response inhibition of neutrophil elastase activity when treated with the AAT polypeptide (sAAT) produced in a CHO expression system to either fresh sAAT or sAAT used after being stored at ambient temperature for one week. [Figure 16] This figure shows a chart illustrating the assay results demonstrating the ability of the AAT polypeptides produced in this disclosure (sAAT, uAAT) and wild-type AAT (referred to as eAAT), when used in mist form, to inhibit neutrophil elastase activity in influenza A-treated neutrophils. [Figure 17] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on reducing infection of human lung epithelial cells by IAV. [Figure 18] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing the number of pulmonary airway white blood cells in mice infected with IAV (3-day exposure model). [Figure 19] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing lung weight in mice infected with IAV (3-day exposure model). [Figure 20] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing body weight loss in mice infected with IAV (5-day exposure model). [Figure 21] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing lung weight in mice infected with IAV (5-day exposure model). [Figure 22]This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing the number of pulmonary white blood cells in bronchoalveolar (BAL) fluid in mice infected with IAV (5-day exposure model). [Figure 23] This figure shows the effect of AAT polypeptide (sAAT) produced in a CHO expression system on treating pneumonia by reducing pulmonary necrosis in mice infected with IAV (5-day exposure model). [Figure 24] This figure illustrates the effect of freeze-dried AAT polypeptide (sAAT) produced in a restored CHO expression system, evaluating its ability to reduce NE activity in vitro. The NET assay for NE expression was performed as described above, and cells were exposed to IAV to increase NE activity. Both concentrations of AAT used eliminated NE activity. [Modes for carrying out the invention]

[0051] definition Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs (e.g., molecular biology, structural biology, cell culture, protein synthesis, protein chemistry, medicinal chemistry, biochemistry).

[0052] Where any prior art document is referenced herein, it will be understood that such reference does not constitute an admission that the document is part of the common general knowledge in the art.

[0053] As used herein, the term “and / or,” for example, “X and / or Y,” should be understood to mean either “X and Y” or “X or Y,” and should be interpreted as providing explicit support for both meanings or either one of them.

[0054] As used herein, the term “approximately” refers to a range of + / - 10% of the specified value unless otherwise specified.

[0055] Throughout this specification, unless otherwise specified or contextually required, references to a single process, composition of a substance, group of processes, or group of compositions of a substance should be interpreted as encompassing both singular and plural (i.e., one or more) aspects of those processes, compositions, groups of processes, or groups of compositions of a substance. Therefore, as used herein, the singular forms "a," "an," and "the" include multiple aspects unless the context clearly indicates otherwise. For example, a reference to "a" includes singular and two or more; a reference to "an" includes singular and two or more; a reference to "the" includes singular and two or more; and so on.

[0056] Unless otherwise noted, terms such as “first,” “second,” etc., are used herein solely for designation and are not intended to impose any sequential, positional, or hierarchical requirements on the items they refer to. Furthermore, references to “second” items do not require or exclude the existence of items with lower numbers (e.g., “first” items) and / or items with higher numbers (e.g., “third” items).

[0057] As used herein, the phrase “at least one of” means, when used with a list of items, that any combination of one or more of the listed items may be used, and that only one of the items in the list may be required. An item may be a specific object, event, or category. In other words, “at least one of” means that any combination or number of items from the list may be used, but not all items in the list may be required. For example, “at least one of item A, item B, and item C” could mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” could mean, for example, two items A, one item B, and ten items C; four items B and seven items C, or several other suitable combinations.

[0058] As used herein, the word “contains” and other forms of the word such as “contains” and “contains (third person singular present)” mean, but not limited to, other additives, ingredients, integers, or processes, for example, and are not intended to exclude them.

[0059] Each embodiment of the disclosure described herein should be applied mutatis mutandis to any other embodiment unless otherwise specified or required by context.

[0060] abbreviation AECOPD: Acute exacerbation of COPD α1-AT α1-antitrypsin AAT α1-antitrypsin arb (arbitrary unit) BAL (Bronchial Alveolar) Bis-ANS 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, diphosphate β-ME 2-mercaptoethanol CD circular dichroism CT (Computed Tomography) CF (Cystic Fibrosis) COPD chronic obstructive pulmonary disease CSE Tobacco Smoke Exposure D degeneration DTT 1,4-Dithiothreitol DMSO (Dimethyl Sulfoxide) eAAT (eAAT) eluted wild-type AAT E.coli Escherichia coli EDTA (Ethylenediaminetetraacetic acid) GCS glucocorticosteroids GndHCl Guanidinium Hydrochloride GVHD (Graft-versus-Host Disease) ΔG Gibbs free energy ΔΔG: Difference in Gibbs free energies HNE (Human Neutrophil Elastase) HPAI (Highly Pathogenic Avian Influenza) IAV (Influenza A virus) IPTG Isopropyl β-D-thiogalactoside MD Molecular Dynamics MET macrophage extracellular trap min MRI (Magnetic Resonance Imaging) N nature NET neutrophil extracellular trap NTHi (Non-Syllable Influenza) OD 600 Optical density at 600 nm PAI-1 Plasminogen Activator Inhibitor-1 PCA principal component analysis PCLS precision lung slices PDB ID: Protein Databank Identifier PEG-3350 / 8000 Polyethylene Glycol 3350 / 800 PFU plaque formation unit rAAT Recombinant Wild-type AAT sAAT Mutant Recombinant AAT RCL reaction center loop ROS (Reactive Oxygen Species) RT, R / T room temperature SI inhibitory stoichiometry s seconds SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Serpine serine protease inhibitors smFRET Single-molecule Förster resonance energy transfer Tm midpoint of thermal denaturation TEMED Tetramethylethylenediamine TLR Toll-like receptor uAAT Mutant Recombinant AAT WCC white blood cell count WT wild type wtAAT wild type AAT

[0061] array In this specification, amino acid sequences are shown with the N-terminus on the left, and are presented across multiple lines, with the N-terminus located in the upper left. Unless otherwise specified, amino acid residues in the sequences are L-amino acids.

[0062] The amino acid sequences listed in this application are indicated using standard letter abbreviations for amino acids.

[0063] The specific sequences provided herein relate to specific embodiments of this disclosure.

[0064] AAT polypeptide This disclosure is based on the remarkable discovery that certain mutations, particularly combinations of mutations, in the AAT sequence can provide improved stability of the resulting polypeptide while retaining functional properties. Until now, stabilizing the native state of AAT without impairing its function has been challenging. The inventors have now identified combinations of mutations that interact to provide improvements in properties such as stability, aggregation resistance, and protein yield. These properties facilitate the easier production and distribution of AAT polypeptides, as well as their ease of use and increased lifespan.

[0065] Therefore, in one embodiment, an alpha-1 antitrypsin (AAT) polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, An AAT polypeptide is provided, in which the amino acid sequence differs from SEQ ID NO: 2 in that it contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

[0066] Human wild-type AAT has the amino acid sequence shown in Sequence ID No. 1, presented in Figure 1. The metastable native conformation of AAT consists of three β-sheets (A-C), nine α-helices (A-I), and a protruding movable loop known as the reaction center loop (RCL). The largest element of the serpin structure is the central β-sheet, β-sheet A, located posterior to Helix-F. The hydrophobic core consists of strands 2-6 (s2B, s3B, s4B, s5B, and s6B) of β-sheet B and Helix-B, while β-sheet B also forms a B / C barrel with β-sheet C. The protruding RCL is important for the AAT inhibition mechanism, acting as a bait for the target protease to bind. The loop is bound from strand 5 of β-sheet A and strand 1 of β-sheet C.

[0067] The AAT polypeptides of this disclosure have at least 90% sequence identity with SEQ ID NO: 2, based on amino acid residues at positions 41-418 of human AAT (SEQ ID NO: 2), and also contain portions of amino acid sequences having at least three mutations (and optionally further amino acid modifications, provided that the AAT polypeptide has a portion of its amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 2) derived from G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I).

[0068] Polypeptides based on amino acid modifications at positions 41-418 have been shown to exhibit improved stability while maintaining functional efficacy, for example, by inhibiting neutrophil elastase activity.

[0069] In some embodiments, the amino acid sequence has 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

[0070] In some embodiments, the amino acid sequence has four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

[0071] In some embodiments, the amino acid sequence is the following group of amino acid substitutions from SEQ ID NO: S276K, T278E, T323E, D325N and K327E, L275F, I324V and M358I, and G99A, T144W, and Y171A It has one, two, or all of them.

[0072] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: S276K, T278E, T323E, D325N, and K327E Includes.

[0073] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: L275F, I324V and M358I Includes.

[0074] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: G99A, T144W, and Y171A Includes.

[0075] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: L275F, S276K, T278E, T323E, I324V, D325N, K327E and M358I Includes.

[0076] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: G99A, T144W, Y171A, S276K, T278E, T323E, D325N, and K327E Includes.

[0077] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: G99A, T144W, Y171A, L275F, I324V and M358I Includes.

[0078] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I Includes.

[0079] In some embodiments, the amino acid sequence includes one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R.

[0080] In some embodiments, the amino acid sequence includes two or more, three or more, four or more, five or more, or each of the following further amino acid substitutions from SEQ ID NO: F35L, T43A, T52A, A54G, S365A, and K371R.

[0081] In some embodiments, the amino acid sequence is further modified from SEQ ID NO: 2 as follows: F35L, T43A, T52A, A54G, S365A, and K371R Includes.

[0082] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: The following groups of amino acid substitutions: S276K, T278E, T323E, D325N and K327E, L275F, I324V, and M358I, and G99A, T144W, and Y171A One, two, or all of them Furthermore, one or more additional amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. Includes.

[0083] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: The following groups of amino acid substitutions: S276K, T278E, T323E, D325N and K327E, L275F, I324V, and M358I, and G99A, T144W, and Y171A One, two, or all of them The molecule also includes two or more, three or more, four or more, five or more, or each of the following further amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R.

[0084] In some embodiments, the amino acid sequence is modified by the following amino acid substitutions from SEQ ID NO: 2. F35L, T43A, T52A, A54G, G99A, T144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, M358I, S365A and K371R Includes.

[0085] The AAT polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, and differs from SEQ ID NO: 2 in that it contains three or more amino acid substitutions as expressly provided herein.

[0086] The AAT polypeptide may optionally contain additional amino acid sequences to the amino acid sequence based on SEQ ID NO: 2. For example, in some embodiments, the AAT polypeptide may contain an additional N-terminal amino acid sequence and / or C-terminal amino acid sequence, in addition to containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2.

[0087] In some embodiments, the AAT polypeptide includes an additional N-terminal amino acid sequence.

[0088] In the modified embodiment, the AAT polypeptide includes an additional C-terminal amino acid sequence.

[0089] In some embodiments, the AAT polypeptide includes a CHO N-terminal sequence.

[0090] In some embodiments, the AAT polypeptide contains an E. coli N-terminal sequence.

[0091] In some embodiments, the AAT polypeptide includes the N-terminal sequence MENLYFQGAAS (SEQ ID NO: 7).

[0092] In some embodiments, the AAT polypeptide contains the N-terminal sequence MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHH (SEQ ID NO: 8).

[0093] In some embodiments, the AAT polypeptide includes the N-terminal sequence MPSSVSWGILLLAGLCCLVPVSLAE (SEQ ID NO: 9).

[0094] In some embodiments, the AAT polypeptide includes a His tag sequence, such as an N-terminal His tag sequence.

[0095] The AAT polypeptide amino acid sequence based on SEQ ID NO: 2 has at least 90% sequence identity with SEQ ID NO: 2.

[0096] In some embodiments, a portion of the AAT polypeptide, which is an amino acid sequence based on SEQ ID NO 2, It contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, and Depending on the circumstances, it may include one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. Furthermore, it does not have amino acid modifications from SEQ ID NO: 2. In other words, some of the AAT polypeptides based on SEQ ID NO: 2 do not have modifications to the amino acid sequence other than those specific positions.

[0097] However, it will be understood that AAT polypeptides containing an amino acid sequence that is a further variant of SEQ ID NO: 2, and having amino acid modifications (e.g., substitutions, deletions, insertions) at other positions, may share the beneficial stability properties observed with the example AAT polypeptide and retain functional activity.

[0098] Therefore, in some embodiments, the AAT polypeptide contains an amino acid sequence based on SEQ ID NO: 2, but has one or more further modifications from the sequence of SEQ ID NO: 2, in addition to any substitutions at the positions defined above.

[0099] In some embodiments, one or more further modifications are one or more substitutions. In some embodiments, one or more further modifications are one or more conservative substitutions.

[0100] A conservative substitution is the replacement of an amino acid residue in a polypeptide with another biologically similar residue. The term “conservative mutation” also includes the use of a substituted amino acid, i.e., an amino acid having one or more atoms replaced by another atom or group, in cases where the polypeptide substantially retains its activity, or where an antibody made against the substituted polypeptide also exhibits an immune response against the unsubstituted polypeptide.

[0101] Typical but non-restrictive conserved substitutions include the substitution of aliphatic amino acids Ala, Val, Leu, and Ile with each other; the exchange of hydroxyl-containing residues Ser and Thr; the exchange of acidic residues Asp and Glu; the exchange of amide-containing residues Asn and Gin; the exchange of basic residues Lys and Arg; the exchange of aromatic residues Phe and Tyr; and the exchange of small amino acids Ala, Ser, Thr, Met, and Gly. Additional conservative substitutions include the substitution of amino acids due to other similar spatial or stereochemical arrangements, e.g., the exchange of Asp with Asn, or Glu with Gin. Non-restrictive examples of conserved amino acid substitutions include: Original residues are replaced by conservative substitutions. Ala Gly, Val, Leu, Ile, Ser, Thr, Met Arg Lys Asn Asp, Gln, His Asp Glu, Asn Cys Ser Gin Asn, His, Lys, Glu Glu Asp, Gln Gly Ala, Ser, Thr, Met His Asn, Gln Ile Ala, Leu, Val, Met Leu Ala, Ile, Val, Met Lys Arg Met Leu, Ile, Ala, Ser, Thr, Gly Phe Leu, Tyr, Trp Ser Thr, Cys, Ala, Met, Gly Thr Ser, Ala, Ser, Met, Gly Trp Tyr, Phe Tyr Trp, Phe Val Ala, Ile, Leu

[0102] Oxidation of methionine in the reaction center loop may reduce the inhibitory activity of AAT.

[0103] In some embodiments, the AAT polypeptide has additional modifications from the sequence of SEQ ID NO: 2, in that the amino acid at position 335 is not Met (i.e., other than the possible modifications considered above at positions 35, 43, 52, 54, 99, 144, 171, 275, 276, 278, 323, 324, 325, 327, 358, 365 and / or 371).

[0104] In some embodiments, the AAT polypeptide has additional modifications from the sequence of SEQ ID NO: 2, in that the amino acid at position 335 is a hydrophobic side chain selected from, for example, Val, Leu, Ile, and Ala. In some embodiments, the amino acid residue is Val. In some embodiments, the amino acid residue is Leu.

[0105] In some embodiments, the AAT polypeptide has an additional modification from the sequence of SEQ ID NO: 2, in that the amino acid at position 335 is glycine.

[0106] In some embodiments, the AAT polypeptide has additional modifications from the sequence of SEQ ID NO: 2, in that the amino acid at position 335 is a charged residue selected from, for example, Glu and Asp. In some embodiments, the amino acid residue is Glu.

[0107] In some embodiments, the AAT polypeptide has additional modifications from the sequence of Sequence ID No. 2, i.e., other than the possible modifications considered above at positions 35, 43, 52, 54, 99, 144, 171, 275, 276, 278, 323, 324, 325, 327, 358, 365 and / or 371, in that the amino acid at position 342 is not Met.

[0108] In some embodiments, the AAT polypeptide has additional modifications from the sequence of SEQ ID NO: 2, in that the amino acid at position 342 is an amino acid having a hydrophobic side chain selected from, for example, Val, Leu, Ile, and Ala. In some embodiments, the amino acid residue is Val. In some embodiments, the amino acid residue is Leu.

[0109] In some embodiments, the AAT polypeptide has an additional modification from the sequence of SEQ ID NO: 2, in that the amino acid at position 342 is glycine.

[0110] In some embodiments, the AAT polypeptide has additional modifications from the sequence of SEQ ID NO: 2, in that the amino acid at position 342 is a charged residue selected from, for example, Glu and Asp. In some embodiments, the amino acid residue is Glu.

[0111] In some embodiments, the only additional modification to the amino acid sequence of the AAT polypeptide from the sequence of Sequence ID No. 2 is that the amino acid at position 335 is not Met and / or the amino acid at position 342 is not Met (i.e., other than the possible modifications considered above at positions 35, 43, 52, 54, 99, 144, 171, 275, 276, 278, 323, 324, 325, 327, 358, 365 and / or 371). For example, the amino acid at position 335 may be Val, Leu or Glu, and the amino acid at position 342 may be Leu.

[0112] In some embodiments, a portion of the AAT polypeptide, which is an amino acid sequence based on SEQ ID NO 2, It contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, and Depending on the circumstances, it may include one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. In addition, it has five or fewer amino acid modifications (e.g., five or fewer conservative substitutions) from SEQ ID NO: 2.

[0113] In some embodiments, It contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, and Depending on the circumstances, it may include one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. In addition, some AAT polypeptides, which are amino acid sequences based on SEQ ID NO: 2, have 10 or fewer amino acid modifications (e.g., 10 or fewer conservative substitutions) from SEQ ID NO: 2.

[0114] In some embodiments, a portion of the AAT polypeptide, which is an amino acid sequence based on SEQ ID NO 2, Three or more amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, Depending on the circumstances, one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R, and Depending on the context, one or more additional amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of M335V, M335L, M335I, M335A, M335E, or M335D, and M342L. It has up to 19 amino acid substitutions from sequence number 2.

[0115] In some embodiments, a portion of the AAT polypeptide, which is an amino acid sequence based on SEQ ID NO: 2, has 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% sequence identity with respect to SEQ ID NO: 2.

[0116] In some embodiments, a portion of the AAT polypeptide, which is an amino acid sequence based on SEQ ID NO: 2, has amino acid modifications of 35, 30 or fewer, 25 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, or 17 or fewer from SEQ ID NO: 2.

[0117] In some embodiments, any modification other than the specific modifications specified above is a conservative substitution.

[0118] In some embodiments, the AAT polypeptide has an amino acid sequence consisting of any of the example AAT polypeptides, for example, the AAT polypeptide has the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0119] In some embodiments, the AAT polypeptide has an amino acid sequence based on the amino acid sequence of SEQ ID NO: 2, They have three or more amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I. They may have one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. They may have additional amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of M335V, M335L, M335I, M335A, M335E, or M335D. They may have additional amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of M342L. They may also have additional N-terminal amino acid sequences and / or C-terminal amino acid sequences. These are derivatives selected from amidation, glycosylation, carbamylation, acylation, sulfonation, phosphorylation, cyclization, lipidation, pegylation, and fusion to another peptide or protein to form a fusion protein. They do not have any other modifications from the amino acid sequence of SEQ ID NO: 2.

[0120] AAT polypeptide derivatives and salts This disclosure encompasses all forms of AAT polypeptides that can produce AAT polypeptides, including derivatives, salts, and / or solvates.

[0121] In some embodiments, the disclosure relates to non-derivative AAT polypeptides, while in other embodiments, the disclosure relates to AAT polypeptides in which the amino acid sequence is derivatized.

[0122] AAT polypeptides may include one or more derivatizations selected from, for example, amidation, glycosylation, carbamylation, acylation, sulfonation, phosphorylation, cyclization, lipidization, pegylation, and fusion to another peptide or protein to form a fusion protein.

[0123] For example, the derivative may include one or more derivatizations selected from amidation, esterification, glycosylation, carbamylation, acylation, sulfonation, phosphorylation, cyclization, lipidization, and PEGylation.

[0124] In some embodiments, the AAT polypeptide includes modifications selected from the group consisting of glycosylation, PEGylation, prenylation, acylation, biotinylation, phosphorylation, and conjugation to a lipid moiety.

[0125] The structure may be modified at random positions within the molecule in some embodiments, or at predetermined positions within the molecule in some other embodiments, and may contain one, two, three, or more bonded chemical moieties.

[0126] For example, pharmaceutically acceptable esters and amides of the AAT polypeptides of this disclosure include ester or amide groups of C1-20 alkyl, C2-20 alkenyl, C5-10 aryl, C5-10 ar-C1-20 alkyl, or amino acid groups bonded at appropriate sites, formed by the reaction of an alkyl, alkenylaryl, aralkyl, or aminoalkyl group containing an alcohol or amino moiety with an acid moiety present in the amino acid sequence of the AAT polypeptide, or by the reaction of an alkyl, alkenylaryl, aralkyl, or aminoalkyl group containing an activated acyl group with an alcohol or amine group present in the amino acid sequence of the AAT polypeptide. Examples of appropriate moieties are hydrophobic substituents having 4-26 carbon atoms. Suitable lipid groups include fatty acids (e.g., lauroyl, palmityl, oleyl, and stearyl (C17H35)) and bile acids (e.g., cholate or deoxycholate).

[0127] AAT polypeptides may be PEGylated, for example. PEGylated derivatives may offer additional advantages such as increased polypeptide solubility, stability, and circulation time, or decreased immunogenicity.

[0128] The chemical moiety for derivatization may also be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, and polyvinyl alcohol. The polymer moiety for derivatization of the AAT polypeptide of this disclosure may have any molecular weight and may be branched or unbranched.

[0129] Salt forms of AAT polypeptides also constitute part of this disclosure. The salts of AAT polypeptides of this disclosure include those that are pharmaceutically acceptable, i.e., suitable for use in pharmaceuticals. Suitable salts according to this disclosure include those formed with organic or inorganic acids or bases.

[0130] Examples of pharmaceutically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, salicylic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and isethionic acid.

[0131] Examples of pharmaceutically acceptable salts with bases include ammonium salts, alkali metal salts such as potassium and sodium salts, alkaline earth metal salts such as calcium and magnesium salts, and organic bases such as dicyclohexylamine and N-methyl-D-glucomine.

[0132] It will be understood by those skilled in the art that many organic compounds can form complexes with the solvent in which they are reacted, or with the solvent in which they are precipitated or crystallized. Such complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” This disclosure also encompasses the solvated forms of AAT polypeptides, solvates of derivatives of compounds, and solvates of salts of derivatives. It will also be understood by those skilled in the art that many organic compounds can exist in various forms, including amorphous materials and / or one or more crystalline forms. Various physical forms of organic compounds are known as polymorphisms. This disclosure also encompasses all such various physical forms of AAT polypeptides, as well as various physical forms of their derivatives and salts.

[0133] Characteristics of AAT polypeptides The AAT polypeptides of this disclosure have activity similar to wild-type alpha-1 antitrypsin, for example, in the inhibition of neutrophil elastase, the downregulation of proteases, and / or in the mediation of anti-inflammatory effects. Therefore, AAT polypeptides are utilized in therapeutic applications, including diseases and disorders associated with AAT deficiency. Neutrophil-driven inflammation is particularly targeted by the action of AAT.

[0134] In some embodiments, the AAT polypeptide has at least 1 / 100 of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 1 / 50 of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 1 / 20 of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 1 / 10 of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 50% of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 80% of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 90% of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 95% of the activity of wild-type human AAT in inhibiting neutrophil elastase, or at least 100% of the activity of wild-type human AAT in inhibiting neutrophil elastase.

[0135] In some embodiments, neutrophil elastase inhibitory activity is determined according to an assay as described in the examples.

[0136] The AAT polypeptides described herein have been shown to have improved thermal stability compared to wild-type human AAT. Therefore, in some embodiments, the AAT polypeptides have improved thermal stability compared to wild-type human AAT.

[0137] The example AAT polypeptides described herein have been shown to have improved refold yields compared to wild-type human AAT, offering an opportunity for higher polypeptide yields. Therefore, in some embodiments, AAT polypeptides have improved refold yields compared to wild-type human AAT. In some embodiments, AAT polypeptides can be obtained with refold rates of at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%.

[0138] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for two weeks (e.g., using the assay described in the examples).

[0139] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for one month (e.g., using the assay described in the examples).

[0140] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for two months (e.g., using the assay described in the examples).

[0141] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for 3 months (e.g., using the assay described in the examples).

[0142] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for 4 months (e.g., using the assay described in the examples).

[0143] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for 5 months (e.g., using the assay described in the examples).

[0144] In some embodiments, the AAT polypeptide retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of its activity in inhibiting neutrophil elastase after storage at 4°C for 6 months (e.g., using the assay described in the Examples).

[0145] Preparation of AAT polypeptide The AAT polypeptides of this disclosure may be produced by recombinant methods known in the art, or similarly, by synthetic methods known in the art.

[0146] For example, AAT polypeptides may be produced by classical peptide synthesis, such as solid-phase peptide synthesis using t-Boc or Fmoc chemistry, or by other well-established techniques; see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis," John Wiley & Sons, 1999; Florencio Zaragoza DOrwald, "Organic Synthesis on Solid Phase," Wiley-VCH Verlag GmbH, 2000; and "Fmoc Solid Phase Peptide Synthesis," edited by WCChan and PDWhite, Oxford University Press, 2000.

[0147] Alternatively, AAT polypeptides can be produced by recombinant methods, i.e., by culturing host cells that contain the DNA sequence encoding the fragment and are capable of expressing the peptide in a suitable nutrient medium under conditions that enable peptide expression. Non-limiting examples of host cells suitable for the expression of these peptides include Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines.

[0148] Therefore, nucleic acid molecules encoding AAT polypeptides as defined herein are provided.

[0149] Furthermore, a vector comprising a nucleic acid sequence encoding an AAT polypeptide as defined herein is provided.

[0150] In some embodiments, the nucleic acid sequence is linked to an expression regulatory sequence suitable for expression in the host cell.

[0151] Furthermore, cells containing nucleic acids as defined herein are provided. In some embodiments, the cells include vectors as defined herein. In some embodiments, the cells are mammalian cells or bacterial cells. In some embodiments, the cells are DH5α, BL21(DE3)pLysS, SG13009, or Rosetta Blue DE3 cells.

[0152] In another embodiment, a method for producing an AAT polypeptide as defined herein, a. Transfecting host cells with a nucleic acid sequence encoding an AAT polypeptide as defined herein, b. Transfected host cells are cultured in cell culture medium and express the AAT polypeptide. c. Recovering AAT polypeptide from cell culture medium and A method including this is provided.

[0153] Specific examples of the method for producing AAT polypeptide according to this disclosure are provided in the examples.

[0154] Pharmaceutical composition In some embodiments, the AAT polypeptides of the Disclosure may be administered on their own if desired, while typically, the AAT polypeptides of the Disclosure are administered in the form of a pharmaceutical composition containing the AAT polypeptide and a pharmaceutically acceptable excipient.

[0155] Accordingly, pharmaceutical compositions comprising an AAT polypeptide as defined herein and a pharmaceutically acceptable carrier are provided.

[0156] Any suitable pharmaceutical composition may be used.

[0157] The pharmaceutical composition may contain, for example, one or more pharmaceutically acceptable carriers, excipients such as solvents or diluents, buffers, pH adjusters, antioxidants, and / or osmotic pressure adjusters.

[0158] Pharmaceutically appropriate compositions of AAT polypeptides can be prepared according to methods known to those skilled in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, edited by S. Frokjaer and L. Hovgaard, Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 31st edition, edited by A. Kibbe, Pharmaceutical Press (2000)). Examples of these formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN®), semi-solid gels, and semi-solid mixtures. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0159] The AAT polypeptides of this disclosure can be prepared in pharmaceutical compositions, for example, in the form of a solution, suspension, or emulsion solid (e.g., powder).

[0160] In some embodiments, the pharmaceutical composition exists in the form of a freeze-dried powder. In some embodiments, the pharmaceutical composition exists in the form of a finely ground powder.

[0161] In some embodiments, the pharmaceutical composition exists in the form of a restorative powder.

[0162] In some embodiments, the pharmaceutical composition exists in liquid form, for example, in solution form.

[0163] AAT polypeptides can be administered by any suitable route, and pharmaceutical compositions formulated to facilitate the delivery of AAT polypeptides by that route may be available.

[0164] In some embodiments, the pharmaceutical composition exists in the form of an injectable formulation, for example, for intravenous injection. In some embodiments, the pharmaceutical composition exists in the form of an inhalable formulation, for example, an inhalation powder or a mist pharmaceutical composition. The pharmaceutical composition may exist in the form of a composition suitable for producing, for example, an aerosolized form of an AAT polypeptide.

[0165] In some embodiments, the pharmaceutical composition exists in the form of an injectable composition, a subcutaneous injection composition, or an intratumor injection composition.

[0166] Pharmaceutical compositions can be prepared by known methods. For example, pharmaceutical compositions can be prepared by dissolving, dispersing, suspending, or emulsifying the above-mentioned polypeptides or salts thereof in a sterile aqueous medium conventionally used for injection. Examples of aqueous media include physiological saline, water for injection, Ringer's solution, dextrose solution, isotonic solutions containing 1-10% human serum albumin, glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants, or similar, if desired.

[0167] When a solid dosage form is used, for example, a solid carrier and / or other excipients may be used.

[0168] Pharmaceutical compositions can be administered in a number of dosage forms, including time-release or sustained-release compositions. Pharmaceutical compositions may exist, for example, in the form of powders, tablets, pills, capsules, microparticles, injections (ampoules), dispersions, or similar. Tablets, pills, capsules, or similar may also contain one or more of the following: binders such as gum or gelatin, excipients, disintegrants, sweeteners, flavorings, additives such as maltose, dextran, sucrose, lactose, mannitol, trehalose, albumin, collagen, gelatin, albumin, preservatives such as sorbic acid, ascorbic acid, alpha-tocopherol, thickeners, buffers, lubricants, etc. Tablets, pills, capsules, or similar may be coated with enteric coatings if desired.

[0169] If desired, the AAT polypeptide may be encapsulated before administration to the subject. The encapsulation process is intended to protect the AAT polypeptide from degradation, thus further preserving the polypeptide's stability and biological activity. For example, the AAT polypeptide may be encapsulated in liposomes or similar materials.

[0170] In some embodiments, the AAT polypeptide may be administered in combination with a further therapeutic agent. In some embodiments, the AAT polypeptide and the further therapeutic agent are contained in the same pharmaceutical composition. In other words, in some embodiments, the pharmaceutical composition comprising the AAT polypeptide and a pharmaceutically acceptable excipient also comprises a further therapeutic agent.

[0171] The AAT polypeptides of this disclosure have good stability properties, meaning they can be stored for long periods and / or at high temperatures while maintaining good activity properties.

[0172] In some embodiments, the pharmaceutical composition retains at least 80%, at least 90%, or at least 95% of its initial neutrophil elastase inhibitory activity after storage at 4°C for 3 months or 6 months (for example, using a neutrophil elastase inhibitory assay as described in the examples).

[0173] Therapeutic applications of AAT polypeptide The AAT polypeptides disclosed herein are used in a variety of therapeutic applications, including the prevention or treatment of AAT deficiency or diseases and disorders associated with AAT deficiency, and various anti-inflammatory effects, particularly on neutrophils.

[0174] Therefore, also provided are AAT polypeptides as defined herein, or pharmaceutical compositions comprising AAT polypeptides, for use as a drug, for example, for use in therapy. Also provided are AAT polypeptides as defined herein, or pharmaceutical compositions comprising AAT polypeptides, for use in the prevention or treatment of AAT deficiency, or diseases or disorders associated with AAT deficiency.

[0175] Furthermore, a method is provided for preventing or treating AAT deficiency, or a disease or disorder associated with AAT deficiency, in a subject, comprising administering to the subject an effective amount of an AAT polypeptide as defined herein, or a pharmaceutical composition as defined herein.

[0176] Furthermore, the use of AAT polypeptides as defined herein is provided for the manufacture of agents for preventing or treating AAT deficiency or diseases or disorders associated with AAT deficiency.

[0177] As used herein, the term “to treat” includes curing a disease or disorder, and reducing or alleviating symptoms associated with a disease or disorder or condition. The term “to treat” also includes slowing the progression of a disease or disorder.

[0178] As used herein, the term “prevention” includes prevention, and includes reducing the likelihood of contracting a disease or disorder or its symptoms.

[0179] The term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans; chimpanzees, and other non-human primates such as apes and monkeys; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish.

[0180] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is a young or child.

[0181] As used herein, the term “effective dose” means a sufficient amount of AAT polypeptide or a pharmaceutical composition containing it that provides a desired therapeutic or preventive effect. The effect includes, but is not limited to, the reduction and / or mitigation of the underlying cause, symptoms, or changes in the underlying physiological pathways that lead to the onset of a disease or condition. In some embodiments, the use of the term “effective dose” refers to a sufficient amount of AAT polypeptide that provides a clinically relevant change in the disease state, the presence or absence of symptoms, or the condition of the disease.

[0182] The term "pharmaceutical" refers to a form of preparation including a formulation, composition, or medical preparation suitable for administration to a target. A pharmaceutical may, for example, include one or more pharmaceutically acceptable excipients. A pharmaceutical may also include, for example, further therapeutic agents.

[0183] AAT polypeptides have been shown to possess potent anti-inflammatory and therapeutic activity, and they are used in the treatment or prevention of numerous diseases or conditions. In particular, the AAT polypeptides of this disclosure have been shown to significantly reduce neutrophil extracellular traps (NETs) and / or macrophage extracellular traps (METs).

[0184] Examples of these modified AAT polypeptides exhibit improved stability and protein folding properties compared to wild-type AATs while retaining beneficial activity, making them desirable for the treatment of various diseases and conditions. For example, these AAT polypeptides may be used in the treatment of diseases or conditions in which the pathogenesis of the disease is related to increased formation of NETs and / or METs.

[0185] Furthermore, this AAT polypeptide may also be useful in treating diseases or conditions associated with an imbalance of pro-inflammatory cytokines or increased inflammation.

[0186] This AAT polypeptide may also be useful in treating diseases or conditions related to or associated with protease activity.

[0187] Therefore, in some embodiments, the disease or disorder is an inflammatory disease, a respiratory disease, cancer, and / or a viral infection.

[0188] In some embodiments, the disease or disorder is selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory diseases, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease (e.g., acute exacerbation of chronic obstructive pulmonary disease), asthma, respiratory diseases associated with air pollution, cancers associated with protease activity (e.g., lung cancer, liver cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

[0189] Neutrophil-driven inflammation is specifically targeted by the action of AAT. In some embodiments, the disease or disorder is an inflammatory disease or disorder, or a neutrophil-associated inflammatory disease or disorder. Further examples of diseases or disorders include cystic fibrosis (CF), bronchitis, liver disease, liver failure, rheumatoid arthritis, transplant rejection, graft-versus-host disease (GVHD), type 1 and type 2 diabetes, and allergic airway diseases.

[0190] The various conditions listed above may be characterized by a neutrophil inflammatory response resistant to standard immunosuppression.

[0191] Experimental data may support the hypothesis that ROS / TLRs are involved in NET / MET trap formation. In particular, NTHi, tobacco smoke exposure (CSE), and influenza A virus all induce ROS production by phagocytic cells such as neutrophils and macrophages.

[0192] Therefore, this disclosure also relates to the administration of the AAT polypeptide for reducing ROS / TLR activity. This disclosure also relates to the administration of the AAT polypeptide in the treatment of diseases or disorders associated with oxidative stress.

[0193] COPD is characterized by chronic inflammation that leads to progressive lung damage. One of the main risk factors for developing COPD is exposure to tobacco smoke (CSE). However, once this inflammatory process is established, COPD tends to persist and progress even after quitting smoking (potential causes of this effect include lingering smoking-related substances, autoimmunity, and infection). The main anti-inflammatory therapies used are glucocorticosteroids (GCS), which are partially effective but have a wide range of side effects.

[0194] Overproduction of proteases and / or deficiencies of inhibitors (e.g., AATs) by innate lung immune cells (e.g., macrophages and neutrophils) leads to protease imbalance. This imbalance usually occurs in the presence of relatively low levels of antiproteases in the lung and is a major mechanism for the development of emphysema. The most important proteases are neutrophil elastase (NE) and macrophage metalloproteinases (MMP) 9 and 12, which are highly pro-inflammatory and chemotactic.

[0195] Acute exacerbations of COPD (AECOPD) are most commonly triggered by bacterial and viral infections, which typically increase lung inflammation. Even with the best available treatments, they frequently result in death and are a major cause of excessive morbidity. AECOPD is the most common cause of hospitalization in Australia. Haemophilus influenzae type 1 (NTHi) is the most common bacterium isolated in COPD patients, especially during exacerbations. Viral infections, such as influenza A virus (IAV), are also a major cause of AECOPD.

[0196] Viral infections can also produce a significant inflammatory response in the subject, particularly in the lungs. These symptoms may be exacerbated by the subject's medical history, such as being a smoker or having a chronic respiratory disease. Examples of viral infections include IAV, including seasonal H1N1 and H3N2 strains, as well as pandemic-potential strains such as HPAI H5N1, H7N9, and H9N2.

[0197] AAT polypeptides are understood to reduce and degrade NETs and METs, leading to reduced inflammation and improvement or cessation of symptoms associated with inflammation or protease imbalance. These AAT polypeptides are also thought to possess potent anti-inflammatory properties that can reduce circulating pro-inflammatory cytokine or chemokine levels in subjects. Exemplary cytokines or chemokines include IL-1β, IL-6, TNF-α, and IFN-γ. These AAT polypeptides are also thought to reduce leukocyte infiltrations in subjects, thereby reducing their destructive pro-inflammatory effects in organs and tissues during chronic and acute diseases.

[0198] This AAT polypeptide is useful in the treatment of AAT deficiency, or diseases or disorders associated with AAT deficiency. One form of AAT deficiency is congenital AAT deficiency. Individuals have two copies of the AAT gene, which allows the liver to produce AAT. However, in certain individuals, one copy of the AAT gene may contain a mutation that affects the individual's ability to produce normal levels of AAT. In some patients, AAT levels may be low enough for the patient to develop ongoing symptoms and health problems. AAT deficiency (AATD) is also called genetic or hereditary emphysema. AATD has been shown to cause chronic lung and liver disease.

[0199] Currently, there is no cure for AATD. Clinical management of AATD involves administering antibiotics for lung infections such as influenza or pneumonia, or managing symptoms with inhaled bronchodilators to alleviate emphysema symptoms such as shortness of breath. AAT supplementation or augmentation therapy can also be used in certain patients. However, natural AAT supplementation or augmentation therapy is expensive considering the instability and production costs of natural AAT. Therefore, many healthcare systems around the world do not cover the patient costs of AAT supplementation or augmentation therapy. This AAT polypeptide has improved stability and higher yield than natural AAT and is therefore desirable.

[0200] This AAT polypeptide is also considered useful in the treatment of cancer. Natural AATs have been shown to inhibit angiogenesis and tumor growth. AAT polypeptides may also be administered in combination with one or more other cancer treatments, such as surgery, radiotherapy, chemoradiotherapy, chemotherapy, cytotoxic agent treatment, antibody therapy, antibody-drug conjugate treatment, and / or cell therapy, such as CAR-T cell therapy.

[0201] Surgery may be used to partially or completely remove a tumor by excision, debulking, or cell reduction surgery. Radiation therapy (or radiation therapy) uses a controlled dose of radiation to kill or damage cancer cells. Radiation therapy is frequently used in combination with chemotherapy, surgery, or the administration of other pharmaceutical compounds. When administered in combination with chemotherapy, the method is called chemoradiation therapy. Radiation therapy may be administered as external beam radiation therapy or internal beam radiation therapy. Radiation therapy may also be administered systemically through the ingestion or injection of radioactive material.

[0202] In some embodiments, administration of an AAT polypeptide or a pharmaceutical composition containing the same achieves one or more of the following: reduction of cytokine levels in the target, reduction of extracellular trap formation in the target organ, reduction of protease activity, and reduction of leukocyte infiltration in the target organ. These can be broadly considered antineutrophil effects.

[0203] Any suitable route or regimen of administration may be used for the AAT polypeptide. For example, the AAT polypeptide may be administered intravenously or by inhalation. In some embodiments, the AAT polypeptide is administered by inhalation, for example, in a mist form.

[0204] Any appropriate dosing regimen for AAT polypeptide may be available. For example, AAT polypeptide may be administered once daily, twice daily, three times daily, four times daily, or as needed. AAT polypeptide may be administered, for example, every two days, every three days, every four days, every five days, every six days, once a week, every two weeks, every three weeks, every four weeks, once a month, or at any other appropriate dosing interval.

[0205] Any appropriate dose of AAT polypeptide may be administered. For example, amounts of AAT polypeptide ranging from 0.001 mg to 1 g / kg of body weight, or 0.01 mg to 250 mg / kg, or 0.01 mg to 50 mg / kg, or 0.01 mg to 10 mg / kg, or 0.01 mg to 1 mg / kg, or 0.1 mg to 250 mg / kg, or 0.1 mg to 50 mg / kg, or 0.1 mg to 10 mg / kg, or 0.1 mg to 1 mg / kg, or 1 mg to 250 mg / kg, or 1 mg to 50 mg / kg, or 1 mg to 10 mg / kg, or up to 1 g / kg, or up to 250 mg / kg, or up to 50 mg / kg, or up to 10 mg / kg, or up to 5 mg / kg, or up to 1 mg / kg, or up to 0.5 mg / kg, or up to 0.1 mg / kg may be administered.

[0206] In some embodiments, the AAT polypeptide of the Disclosure may be administered as a single therapeutic agent, while in other embodiments, the AAT polypeptide of the Disclosure may be administered together with one or more further therapeutic agents.

[0207] Therefore, in some embodiments, the AAT polypeptide or pharmaceutical composition is administered in combination with a further therapeutic agent.

[0208] AAT polypeptide and further therapeutic agents may be administered according to any dosing regimen suitable for the treatment or prevention of the associated disease or disorder. For example, AAT polypeptide and further therapeutic agents may be administered simultaneously (e.g., AAT polypeptide and further therapeutic agents may be administered in the same composition or via different compositions administered simultaneously), sequentially (e.g., one drug is administered first, then the other), or separately (e.g., different drugs may be administered at different times and / or on different days).

[0209] Further examples of therapeutic agents include those for the treatment of inflammatory diseases, respiratory diseases, cancer, and / or viral infections. In some embodiments, further therapeutic agents are those for the treatment of diseases or injuries selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory diseases, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease (e.g., acute exacerbation of chronic obstructive pulmonary disease), asthma, respiratory diseases associated with air pollution, cancers associated with protease activity (e.g., lung cancer, liver cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

[0210] Further examples of therapeutic agents include steroid activators, NSAIDs, antiviral agents, antifibrotic agents, and anticancer agents.

[0211] Furthermore, this specification provides a kit comprising a) the AAT polypeptide according to this disclosure and b) a further therapeutic agent.

[0212] Furthermore, this specification provides a kit comprising: a) a first pharmaceutical composition comprising the AAT polypeptide according to this disclosure and a pharmaceutically acceptable carrier and / or excipient; and b) a second pharmaceutical composition comprising a further therapeutic agent and a pharmaceutically acceptable carrier and / or excipient.

[0213] This disclosure is further illustrated by the following non-limiting embodiments. [Examples]

[0214] material and method Determination of AAT polypeptide inhibitory activity - Assay buffer: 50mM Tris-HCl, 150mM NaCl, 0.2% (v / v) PEG 8000, pH 7.4 - Protease: Trypsin (1 mM HCl), HNE (50 mM sodium acetate, 150 mM NaCl, pH 5.5) - Chromogenic substrate for trypsin: Na-benzoyl-L-arginine 4-nitroanilide hydrochloride (in DMSO) -Chromogenic substrate for HNE: N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (in DMSO) - BMG FLUOstar Optima Plate Reader (405nm)

[0215] Inhibitory stoichiometry (SI) Protocol: The inhibitory stoichiometry (SI) of each AAT polypeptide against proteases was performed as previously described (Horvath et al.). Various concentrations of AAT polypeptides were incubated with a constant concentration of protease (resulting in a range of 0–2:1) in assay buffer at 37°C for 30 minutes to form AAT polypeptide:protease complexes. Residual protease activity was measured using an Optima plate reader set to 405 nm after adding the target substrate (200 μM) for 1 hour. The change in absorbance from substrate addition to the final reading after 1 hour was plotted as the percentage of active protease relative to the serpine:protease ratio. The data were normalized using 100% complete protease activity (AAT polypeptide:protease 0:1) and fitted using linear regression, where the interaction with the X-axis determined the SI.

[0216] Determination of refolding and inhibitory stoichiometry - Unfolding buffer: 6M GndHCl, 50mM Tris-HCl, 150mM NaCl pH 8.0 - Refolding buffer: 50mM Tris-HCl, 150mM NaCl pH 8.0

[0217] Protocol: The AAT polypeptide was unfolded in an unfolding buffer consisting of 6 M GndHCl for 2 hours and then refolded by dilution for an additional 2 hours. Any aggregates formed were pelleted by centrifugation (tabletop centrifuge, 16,000×g at 4°C for 5 minutes), and the refolded sample was dialyzed against the refolding buffer to remove residual GndHCl. SI was performed as described above.

[0218] AAT Polypeptide: Protease Complex SDS-PAGE Gel - 10% SDS-PAGE Gel - 6× Laemmli SDS Loading Dye

[0219] Protocol: Different concentrations of the AAT polypeptide (resulting in ratios of 1:1 and 2:1) were incubated with a fixed concentration of protease at 37°C for 30 minutes to form the AAT polypeptide:protease complex. The reaction was stopped after 30 minutes by the addition of 6× reducing sample buffer and quenching of the sample on ice. The samples were subjected to SDS-PAGE using a 10% acrylamide gel as described above. The gel was stained with Coomassie Blue and destained to observe the formation of any SDS-stable AAT polypeptide:protease complexes.

[0220] Biophysical Analysis of AAT Polypeptide Circular Dichroism (CD) - Protein Buffer: 1× Phosphate Buffered Saline (1× PBS) (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)

[0221] All circular dichroism techniques were performed using a Jasco J-815 circular dichroism spectrometer at a protein concentration of 0.2 mg / ml in 1× PBS, pH 7.4, using a quartz cell with a path length of 0.1 cm.

[0222] Spectral Scan The secondary structure of the AAT polypeptide was observed using far-ultraviolet spectroscopy (195–250 nm) at a scanning speed of 100 nm / min. The concentration of the AAT polypeptide was 0.2 mg / ml, and the temperature was kept constant at 20°C.

[0223] Thermal denaturation Thermal denaturation was performed by increasing the temperature from 25°C to 100°C at a rate of 1°C / min (unless otherwise specified), and the signal change was recorded at 222 nm. The AAT polypeptide concentration remained at 0.2 mg / ml. The sample was cooled from 100°C to 25°C, and whether any thermal refolding occurred was observed. The midpoint of the transition (T) was determined by fitting the data using a Boltzmann S-curve for both melting and back-melting / cooling denaturation experiments. m ) was obtained.

[0224] For AAT polypeptides that did not undergo unfolding transition upon heating to 100°C, 2M GndHCl was added to aid unfolding. The samples were again subjected to temperature increases from 25°C to 100°C with 2M GndHCl to determine the midpoint of the transition.

[0225] Determination of thermodynamic properties using thermal fusion The thermodynamic properties of the AAT polypeptide were determined using the unfolding transition gradient of thermal melting. First, the ellipticity signal was normalized to create a Y-axis for the "fraction of unfolded material" against temperature. The "fraction of unfolded material" is given by the following equation:

[0226]

number

[0227] (In the formula, f u It was converted to Keq using (where is the fraction of the unfolded part). Then K eq logK eqwas converted. The temperature was converted to Kelvin (°C + 273.15) and then to 1 / temperature (K). Next, logK eq was plotted against the corresponding 1 / temperature (K) and a van't Hoff analysis was performed (John et al., Greenfield et al., Matthews et al.). The resulting plot was fitted to a linear equation and the change in entropy ΔS was determined from the y-intercept using the gas constant R (8.314 J / K / mol) and the following equation:

[0228] [Number]

[0229] The change in enthalpy ΔH was calculated using the slope of the line, the gas constant R, and the equation:

[0230] [Number]

[0231] The Gibbs free energy determined by thermal denaturation was calculated using the Gibbs free energy equation at room temperature (25°C): ΔG = ΔH - TΔS

[0232] The change in Gibbs free energy (ΔΔG) between WT and an exemplary AAT polypeptide was calculated using the equation: ΔΔG = ΔG WT - ΔG (graft)

[0233] Analysis of protein refolding by gel filtration - AKTA FPLC (GE Healthcare) - Superdex 200 increase 10 / 300 GL (GE Healthcare) - Protein / refolding buffer: 1× Tris-buffered saline (1× TBS) (150 mM NaCl, 25 mM Tris-HCl, pH 7.4) - Unfolding buffer: 8M GndHCl (in 1 x TBS at pH 7.4)

[0234] Protocol: Refold analysis was performed by comparing the absorbance peaks (at 280 nm) of the native and refolded proteins using a Superdex 200 10 / 300 column at a final concentration of 2 μM. For refolding, the AAT polypeptide was unfolded in 5 M GndHCl for 1.5 hours, and then refolded by diluting the sample 10-fold with refolding buffer until the final protein concentration reached 2 μM. The refolded sample was centrifuged to remove any aggregates, and a total volume of 500 μl was loaded onto the column. The yield was analyzed by comparing the peak absorbances between the native and refolded AAT polypeptides and normalizing the refolded peaks relative to the peak absorbance of wtAAT (where the peak absorbance of the native protein is 100%).

[0235] ANS Unfolding -Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) - Natural buffer solution: 1 × phosphate-buffered saline (1 × PBS) -8M GndHCl (in 1x PBS, pH 7.4) -4,4'-Dianilino-1,1'-Binaphthyl-5,5'-Disulfonic Acid Diphosphate (Bis-ANS)

[0236] Protocol: Bis-ANS unfolding experiments were performed using a method similar to equilibrium unfolding, but in the presence of Bis-ANS. Protein samples (2 μM concentration) were incubated for at least 3 hours in various concentrations of GndHCl (ranging from 0 to 6 M) and in a 5 molar excess of Bis-ANS (final concentration 10 μM). Bis-ANS fluorescence readings were obtained from a cuvette with a path length of 1 cm, with an excitation wavelength of 390 nm and emission detected at 400–700 nm (5 nm slit for both excitation and emission wavelengths). Peak fluorescence was plotted against the corresponding GndHCl concentration, and chemical unfolding intermediates were analyzed.

[0237] Validation experiments were generally expressed as the mean and the standard error of the mean (or median), and standard parametric / nonparametric analysis was used as appropriate.

[0238] Effects of AAT polypeptide in primary human cells (in vitro) Methods have been developed to study extracellular traps as inflammation in vitro and in vivo. See, for example, King et al., PLoS One 2015;10:e0120371; King et al., Sci Rep 2017;7;12128; Dousha et al., J Vis Exp 2021; King et al., ERJ Open Res, 2021, 7.

[0239] King et al., in ERJ Open Res, obtained wild-type AAT (wtAAT) from human serum by column isolation. Neutrophils derived from peripheral blood or lung macrophages derived from bronchoscopy were infected with bacteria (NTHi) or influenza, and extracellular traps were induced using protease co-expression.

[0240] Paired blood samples for bronchoalveolar (BAL) macrophages and neutrophils were obtained from subjects who underwent bronchoscopy as part of standard clinical care. Non-smoking subjects without detectable lung disease being studied for chronic cough were selected.

[0241] Human BAL macrophages and blood neutrophils (isolated from peripheral blood by centrifugation) on coverslips were cultured in 24-well plates in culture medium at a rate of 10 per well. 5 Individual cells were infected with various IAV strains (in parallel with an uninfected control group).

[0242] The following parameters were measured:

[0243] MET / NET expression: These were analyzed using confocal microscopy in multiple ways: 1) extracellular chromatin with co-expression of protease / histone / PAD2 / 4, 2) % of cells with enlarged nuclei with extracellular chromatin, 3) % of extracellular DNA staining for SYTOX, and 4) indirectly by the presence of extracellular DNA, and using staining with eFluor / Capase-3 to confirm cell viability. Trap expression was analyzed using standard software including IMARIS and Fuji Image J.

[0244] NE activity: In neutrophils, measurements were taken using a standard chromogenic assay, starting at 30-minute intervals and continuing for up to 4 hours.

[0245] Generating animal models: Mice were either intranasally infected with IAV (1000 PFU of H3N2 for mild disease and 50 PFU of H1N1 for severe disease) or left uninfected as controls. In initial experiments, mice were infected on the same day as IAV infection. However, to mimic clinical practice with more detailed experiments following one day after infection, mice were treated with a nebulized medication delivered daily intranasally using recombinant AAT (Rx): Mice were sacrificed on days 4 and 11 to cover a range of inflammatory changes. The peak of the inflammatory response typically appears on day 3 post-infection.

[0246] Storage effects on AAT polypeptide The test was performed using human neutrophils collected from healthy donors, seeded at 100,000 neutrophils per well in a 96-well plate. Except for the control group, all samples were inoculated with 10 MOI H1N1 (Brazil 78) influenza (+ / - treated), incubated at 37°C for 1 hour, then NE substrate was added, and the cells were incubated at 37°C for another 1 hour. Readouts were performed at 405 nm, and background readings were performed at 550 nm.

[0247] Treatment group: wtAAT(R / T) derived from human plasma - taken directly from storage at -20°C and stored at room temperature for up to 3 days. uAAT (approximately 2 weeks) - Store at 4°C for 2 weeks. uAAT (approximately 5 months) - Stored at 4°C for approximately 5 months.

[0248] To prepare dose-response curves, the effects of recombinant AAT at various concentrations were tested against 0.5 μg of porcine neutrophil elastase (Sigma), and activity was evaluated using the same NE substrate and absorbance settings as described above.

[0249] Example 1: Preparation of AAT polypeptide Figure 1 provides the amino acid sequences of recombinant AAT polypeptides AAT (SEQ ID NO: 3 and SEQ ID NO: 4) and uAAT (SEQ ID NO: 5 and SEQ ID NO: 6), wild-type human AAT (SEQ ID NO: 1), and the sequence of wild-type human AAT (SEQ ID NO: 2) on which the AAT polypeptides of this disclosure are based.

[0250] Wild-type human AAT was purified from human plasma or generated in CHO or E. coli cell lines.

[0251] Recombinant AAT polypeptides, AAT (SEQ ID NO: 3) and uAAT (SEQ ID NO: 5), were prepared as described below.

[0252] bacterial cell system

[0253] [Table 1]

[0254] DNA transformation into E. coli cells vector: The vectors used to express recombinant wtAAT were pLIC-His and pQE-31 (Qiagen). Gene sequences for two exemplary alpha-1 antitrypsin AAT polypeptides—sAAT and uAAT—were synthesized and cloned into the pQE-31 vector using Genscript (USA).

[0255] [Table 2]

[0256] Buffer: -LB medium: Yeast extract (5g / L), peptone (tryptone, 10g / L), NaCl (10g / L) -LB Agar: Yeast extract (5g / L), peptone (tryptone, 10g / L), NaCl (10g / L), 1.5% agar - Antibiotics: Ampicillin (100 mg / ml), chloramphenicol (34 mg / ml), and kanamycin (50 mg / ml).

[0257] Protocol: Thaw 20 μl of competent cells, mix with 1 μl of DNA, and incubate on ice for 30 minutes. Heat shock the competent cells at 42°C for 45 seconds, then incubate on ice for 2 minutes. Add 100 μl of LB medium to the cells and harvest them at 37°C for 1 hour. Plate the cells on LB agar (with appropriate antibiotics) and incubate overnight at 37°C.

[0258] Protein expression Buffer: -2×YT medium: Yeast extract (10g / L), peptone (tryptone, 16g / L), NaCl (5g / L) - 2×YT agar: Yeast extract (10 g / L), peptone (tryptone, 16 g / L), NaCl (5 g / L), agar (1.5%) - Antibiotics: Ampicillin (100 mg / ml), chloramphenicol (34 mg / ml), kanamycin (50 mg / ml) and tetracycline (12.5 mg / ml) - Isopropyl-β-D-thiogalactoside (IPTG): 1 M

[0259] Protocol: A single colony from transformation (according to the above protocol) was inoculated into a 10-ml overnight culture of 2×YT medium (with appropriate antibiotics) and incubated overnight at 37°C with constant shaking. This overnight culture was inoculated into 1 liter of 2×YT and then incubated at 37°C with shaking until the cells reached an OD 600 of 0.6. Protein expression was induced with IPTG to a final concentration of 1 mM, and the cells were allowed to express the protein at 37°C for 4 hours. After the specified protein expression time frame, the cells were harvested by centrifugation (4,000×g, 15 minutes at 4°C), and the cell pellet was stored at -20°C.

[0260] Protein purification Wild-type α1-antitrypsin (wtAAT), sAAT, and uAAT were expressed in both soluble and insoluble forms in E. coli depending on the type of vector used for protein expression. wtAAT in the pQE-31 vector was expressed in the soluble fraction / cytoplasm of bacteria (Pearce et al.), while AAT expressed in the pLIC-His vector was expressed insoluble in inclusion bodies. sAAT and uAAT were expressed in inclusion bodies despite being cloned into the DNA sequence of the pQE-31 vector.

[0261] Soluble expressed protein: Cell lysis buffer: 25 mM NaH2PO4, 500 mM NaCl, 1 mM β-ME, 10 mM imidazole, pH 8.0 Nickel-NTA (Ni-NTA) affinity chromatography (natural conditions): -Loose nickel-NTA resin (Qiagen) -Ni-NTA wash buffer: 25mM NaH2PO4, 150mM NaCl, 1mM β-ME, 10mM imidazole, pH 8.0 -Ni-NTA elution buffer: 25mM NaH2PO4, 150mM NaCl, 1mM β-ME, 250mM imidazole, pH 8.0

[0262] Ion exchange chromatography: - AKTA FPLC (GE Healthcare) - HiTrap Q FF anion chromatography column (GE Healthcare) -Dilution buffer: 50mM Tris-HCl pH8.0 -Anion buffer A: 50mM Tris-HCl, 50mM NaCl, 1mM EDTA, pH8.0 -Anion buffer B: 50mM Tris-HCl, 1M NaCl, 1mM EDTA, pH8.0

[0263] Protocol: Cell pellets containing soluble AAT were resuspended in cell lysis buffer and incubated on ice for 20 minutes. After disrupting the cells on ice by sonication (6 × 25 seconds on / 35 seconds off), the cells were centrifuged at 48,000 × g for 20 minutes (4°C) and the cell debris was pelleted. The soluble fraction was loaded onto pre-equilibriumized loose Ni-NTA resin and incubated at 4°C for 1 hour to induce batch binding. Any unbound proteins were eluted and collected, while loose-bound proteins were eluted with Ni-NTA wash buffer. Soluble AAT was eluted into 1 ml fractions using Ni-NTA elution buffer. Fractions containing proteins (when detected by a protein:Bradford reagent 1:10 assay) were pooled and diluted in a 1:1 ratio with dilution buffer. The diluted samples were loaded onto pre-equilibriumized HiTrap Q FF anion exchange chromatography columns. Ion-exchange chromatography was used to separate the various conformations that serpines can adopt (i.e., separating the active native conformation from the inactive subclinical conformation and aggregate conformation). Any non-binding proteins were collected, and the native AAT was eluted by increasing the sodium chloride (NaCl) concentration. The protein-containing fractions were passed through SDS-PAGE to ensure purity, and their inhibitory activity against trypsin was tested. The pure active proteins were rapidly frozen and stored at -80°C for long-term storage, or stored at 4°C for use.

[0264] Insoluble expression proteins: Cell lysis buffer: 50mM NaH2PO4, 300mM NaCl, 10mM imidazole pH8.0 Unfolding Inclusion Body Buffer: 8M urea, 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, 10mM β-ME, pH 8.0 Nickel-NTA (Ni-NTA) affinity chromatography (under modified conditions): -Loose nickel-NTA resin (Qiagen) -Ni-NTA wash buffer: 8M urea, 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, 10mM β-ME, pH 8.0 -Ni-NTA elution buffer: 8M urea, 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 10mM β-ME, pH 8.0 Refolding buffer: 50mM Tris-HCl, 50mM NaCl, 5mM DTT, pH 8.0 Ion exchange chromatography: - AKTA FPLC (GE Healthcare) - HiTrap Q FF anion chromatography column (GE Healthcare) -Anion buffer A: 50mM Tris-HCl, 50mM NaCl, 1mM EDTA pH8.0 -Anion buffer B: 50mM Tris-HCl, 1M NaCl, 1mM EDTA pH8.0

[0265] Protocol: Cell pellets containing insoluble AAT polypeptide (AAT expressed in inclusion bodies) were resuspended in cell lysis buffer and incubated on ice for 20 minutes. Cells were disrupted on ice by sonication (30 seconds on / off), and inclusion bodies were recovered by centrifugation (48,000 × g, 4°C for 20 minutes). Since the protein has a 6 × His tag, inclusion body preparation was unnecessary. Instead, nickel-NTA (Ni-NTA) affinity chromatography was performed under denaturing conditions to partially purify the AAT polypeptide from the inclusion bodies. The AAT polypeptide in the inclusion bodies was resuspended in unfolding inclusion body buffer by continuous stirring with a magnetic stirrer at room temperature (approximately 21°C) for 2 hours. Any proteins that were not resuspended were pelletized by centrifugation (35,000 × g, 4°C for 20 minutes). The soluble components were filtered through a 0.8 μm filter, loaded onto pre-equalized loose Ni-NTA resin, and batch-bound at 4°C for 1 hour with agitation. Any unbound proteins were collected, while loosely bound proteins were eluted with Ni-NTA washing solution. α1-AT was batch-eluted with Ni-NTA elution buffer. To ensure the presence of proteins in the eluted samples, a protein:Bradford reagent 1:10 assay was performed.

[0266] The eluted AAT polypeptide was refolded by a 1:200 dilution in refolding buffer. The refolding buffer contained DTT to reduce cysteine ​​residues and prevent disulfide-driven aggregation. Refolding was performed overnight at 4°C with constant stirring to minimize concentration-dependent aggregation. Any aggregates that occurred were removed by filtration of the refolding buffer through a 0.22 μm filter, and the refolds were loaded onto a pre-equilibrated HiTrap Q FF anion chromatography column. Any unbound proteins were collected. Proteins detected in the fraction were run on SDS-PAGE to confirm purity and tested for inhibitory activity against trypsin. The pure active AAT polypeptide was pooled and either rapidly frozen and stored at -80°C for long-term storage, or stored at 4°C for use.

[0267] Protein buffer exchange - AKTA FPLC (GE Healthcare) - HiTrap desalting column (GE Healthcare)

[0268] Protocol: A HiTrap desalting column was used to exchange all proteins for a specific buffer. The column was pre-equilibrated with the target buffer, and proteins were loaded onto the HiTrap desalting column in 1 ml volumes using AKTA FPLC. The protein-containing fractions were pooled and stored at 4°C for use.

[0269] Determination of protein concentration The concentration of each protein sample was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific), and A 280 Absorbance was measured using the following method. The extinction coefficient of each protein is A 280 This is included when measuring and was calculated using the ExPASy ProtParam online tool (https: / / web.expasy.org / protparam / ).

[0270] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE gels were run at the end of each protein purification procedure to verify protein purity. The gels were subjected to Western blotting only during trial expression to determine whether the target protein was present in the soluble (cytoplasmic) or insoluble (inclusion body) fractions within bacterial cells.

[0271] reagent: -37.5% (40%) acrylamide solution -4x resolution buffer: 0.5M Tris-HCl, 0.4% (w / v) SDS, pH 6.8 -4x stacking buffer: 1.5M Tris-HCl, 0.4% (w / v) SDS, pH 8.8 -ddH2O -20% (w / v) APS -TEMED -1×SDS running buffer: 25mM Tris base, 192mM glycine, 0.1% (w / v) SDS -6×Laemmli SDS sample loading dye: 375mM Tris-HCl, 9% SDS, 50% glycerol, 60mM DTT, and 0.03% bromophenol blue - Precision Plus Protein Dual-Color Standard (Bio-Rad) - Staining solution: 50% (v / v) ddH2O, 40% (v / v) methanol, 10% (v / v) acetic acid, 0.1% (w / v) Coomassie Brilliant Blue G-250 - Destaining solution: 50% (v / v) ddH2O, 40% (v / v) methanol, 10% (v / v) acetic acid -Bio-Rad Mini-PROTEAN Tetra System

[0272] SDS-PAGE gels were constructed using a 10% splitting gel and a 4% stacking gel. The gels were constructed as follows:

[0273] [Table 3]

[0274] Protocol: Each sample to be subjected to electrophoresis was diluted with 6× reducing sample dye, boiled at 90°C for 5 minutes, and then loaded into the stacking wells of an SDS-PAGE gel. The gel was run at 250V for 30 minutes, or until the front of the dye reached the bottom of the gel, whichever came first. The gel was stained with Coomassie blue protein stain or subjected to Western blotting. If the gel was to be stained with Coomassie blue, Coomassie blue protein stain was added to the gel and incubated with agitation for 30 minutes (at room temperature). Subsequently, the stain was removed and a destaining agent was added until the gel background was destained.

[0275] Western blot reagent: -TBS-T: 1x Tris-buffered saline (150mM NaCl, 25mM Tris-HCl, pH7.4), 0.1% (v / v) Tween 20 -Blocking buffer: TBS-T, 5% (w / v) skim milk powder -Transcription buffer: 25 mM Tris base, 190 mM glycine, 15% methanol - Anti-His HRP labeled mouse monoclonal IgG - ECL Western blot detection reagent (Amersham) - Fuki Medical X-ray Film (Fujifilm)

[0276] Protocol: After performing SDS-PAGE gel electrophoresis, the proteins were transferred to a methanol-immersed PVDF membrane in transfer buffer at 100V for 1 hour using a Bio-Rad transfer system. The membrane containing the transferred proteins was blocked with blocking buffer for 1.5 hours, then the blocking buffer was removed, and anti-His HRP-labeled antibody (1:10,000) in TBST was added and left for another hour. After antibody binding, the PVDF membrane was washed four times with TBST for 3 minutes each. ECL Western blot reagent was mixed and added to the membrane, and the membrane was exposed to X-ray film for 30 seconds, 1 minute, and 30 minutes in a light-shielding cassette. The film was developed with X-ray developer.

[0277] Production of AAT polypeptide by CHO cell expression A total of 3× alpha-1-antitrypsin (AAT) protein variants were expressed in ExpiCHO cells for the purpose of glycan profiling. After purification by IMAC affinity, the His-tagged TEV cleavage was performed.

[0278] overview Transient CHO expression leads to protein production of 3×AAT protein. • Protein DNA (with an N-terminal His tag) was synthesized using ThermoFisher Generator and cloned into a mammalian expression vector (pcDNA3.4). • Purification of sterile DNA • Transient transfection of ExpiCHO cells in 0.5L scale. • Purification using IMAC, followed by desalting and TEV cutting.

[0279] Expression vector DNA synthesis, along with cloning into a mammalian expression vector, was outsourced. The target protein sequence design and associated biophysical properties are summarized in the table below, and the sequence is shown in Figure 1. A mammalian Ig-derived leader sequence was added to the sequence design along with the 5' Kozak sequence. DNA synthesis, along with cloning into an expression vector (pcDNA 3.4 TOPO) and preparation of transfection-grade DNA, was outsourced to Geneart. Codon optimization for expression in Chinese hamsters (Cricetulus griseus) was applied. The plasmids were assigned the internal names NBF6401, NBF6402, and NBF6403 for uAAT, sAAT, and rAAT, respectively. uAAT and sAAT are the mutant recombinant AAT polypeptides according to this disclosure. rAAT is the recombinant wild-type AAT.

[0280] [Table 4]

[0281] DNA preparation Transformation AAT expression plasmids were used to transform chemically competent E. coli cells (prepared in-house from Sigma Sig10 chemically competent cells) by heat shock treatment. Briefly, 100 ng of DNA was added to thawed competent cells and cooled on ice for 30 minutes. Next, the cells were heat-shocked at 42°C for 45 seconds, followed by incubation on ice for 2 minutes. Subsequently, 250 μL of Luria Bertani medium was added, and the cells were incubated at 37°C for 1 hour before being harvested. The cultures were then transferred to a flask containing 400 mL of LB medium supplemented with 100 μg / μL ampicillin and grown overnight in a shaking incubator at 37°C and 200 rpm.

[0282] DNA extraction and purification Plasmid DNA was purified using the NucleoBond XtraMidi kit (Macherey-Nagel Cat.740410.100, Lot:21011007) according to the manufacturer's instructions. DNA cleanup with 70% v / v ethanol (Merck Absolute Ethanol #1.00983.2511, Invitrogen UltraPure Distilled Water #10977-015, Lot:2323159) was performed under sterile conditions to prepare the plasmid for transfection. The DNA yield and purity obtained from the extraction are shown in the table below.

[0283] [Table 5]

[0284] Transient transfection Thermo Fisher Scientific's ExpiCHO® cells were thawed and grown in ExpiCHO expression medium (Thermo Fisher Scientific #A29100-01, Lot:2455144) in a 37°C shaking incubator at 130 rpm with a relative humidity of 81% and CO2 of 7.5%, according to the manufacturer's recommendations.

[0285] For each AAT variant, as outlined in the table below, ExpiFectamine CHO reagent (Thermo Fisher Scientific #100033021, Lot:2418917) was transfected into a starting volume of 2 × 211 mL of ExpiCHO culture. Cells were cultured in an Erlenmeyer flask (Corning #431147). ExpiFectamine® CHO enhancer (Thermo Fisher Scientific #100033018, Lot:2430255) and ExpiCHO Feed (Thermo Fisher Scientific #A29101-02, Lot:2378107) were supplied to the culture 22 hours after transfection, bringing the final culture volume to approximately 2 × 280 mL. The culture was then maintained in a 32°C shaking incubator at 130 rpm with a relative humidity of 81% and CO2 of 7.5%. The culture was collected by centrifugation on day 5 after transfection. The supernatant was further clarified by passing it through a 0.22 μm PES sterile filter unit vacuum (Nalgene #567-0020, Lot: 1266567, Nalgene #595-4520, Lot: 1333032) and stored at -80°C until purified.

[0286] [Table 6]

[0287] purification Immobilized metal affinity chromatography (IMAC) affinity Initial purification was performed on only 30 mL of the 500 mL of recovered material. The remaining recovered material will be stored at -80°C for future purification.

[0288] All purification steps were performed at room temperature using an AKTA Pure 25 chromatography system (GE Healthcare) at a flow rate of 2 mL / min.

[0289] The cell culture recovery (30 mL for each AAT variant) was first filtered using a 0.22 μm bottle-top filter (Thermo Scientific), and then purified using a 1 × 1 mL Ni Sepharose column (HisTrap Excel-Cytiva).

[0290] For each recovered sample, the column was washed and disinfected by contact with 0.5 M NaOH for 60 minutes prior to purification, and then equilibrated with DPBS (saline PBS) containing 500 mM NaCl. The column was first equilibrated with 5 CV of binding buffer (pH 7.4), and then the filtered culture supernatant was loaded. The column was washed with 5 CV of saline DPBS (supplemented with 25 mM imidazole), and the protein was eluted from Ni-Sepharose using saline DPBS (supplemented with 500 mM imidazole). The eluate was frozen at -20°C before the desalting step. The thawed material was buffered with 50 mM Tris (pH 8.0) and 150 mM NaCl using diafiltration via a spin filter (Amicon Ultra 10kDa, Merck, UFC 901024). A list of all buffers used is shown in the table below.

[0291] [Table 7]

[0292] The protein concentration of the final product was determined by UV / VIS absorbance at 280 nm (Thermo Scientific, NanoDrop 2000 UV-Vis spectrophotometer).

[0293] TEV cutting The N-terminal His tag of each purified AAT protein was removed using TEV enzyme cleavage (NEB P81125, Lot No. E2103001). Briefly, purified proteins (approximately 6.5 mL each at approximately 1.7 mg / mL) were cleaved overnight at +4°C for 18 hours with gentle stirring in the presence of 50 mM Tris (pH 7.5), 0.5 mM EDTA, 1 mM DTT, and 150 mM NaCl, using 0.4 U of enzyme per μg of protein. Initial small-scale TEV cleavage was performed to optimize the enzyme conditions (incubation temperature and enzyme amount) (data not shown).

[0294] The IMAC eluate (high imidazole content) was frozen during the material processing.

[0295] For large-scale analysis, approximately 1 mg of each AAT variant was cleaved using TEV. The reaction mixture was then passed through an IMAC affinity column to capture the His-tagged TEV enzyme, allowing for the collection of untagged AAT material during flow-through (chromatogram not shown). Cleavage efficiency and structural integrity of the purified material were verified by SDS-PAGE (stain-free, anti-His Western blot) and SE-UPLC. Final protein details, including protein concentration, final volume, and yield, are recorded in the table below.

[0296] [Table 8]

[0297] Analysis of in-process samples SDS-PAGE analysis was performed under reducing conditions only, using 4-15% Mini-PROTEAN® TGX Stain-Free Protein Gels (Biorad, Catalog No.: 4568095), flowing at 200V for 35 minutes. A total of 5 μL of protein ladder was loaded to evaluate accurate separation (Biorad Precision Plus unstained, Catalog No.: 1610363).

[0298] Next, the same SDS-PAGE gel was transferred to a PVDF membrane and blocked overnight at 4°C with PBS-T (1×PBS supplemented with 0.05% Tween-20 and 2% skim milk). The membrane was then washed three times in PBS-T for 15 minutes each, followed by probing with an Anti-His-HRP conjugate antibody (Miltenyi Biotec, Catalog No.: 130-092-785) at a 1:10,000 dilution at room temperature for 1.5 hours. Finally, the membrane was washed three times in PBS-T for 15 minutes each and analyzed using a Novex chemiluminescence substrate kit (ThermoFisher, Catalog No.: WP20005). Subsequently, the membrane was imaged using the Bio-Rad Chemi-Doc™ XRS+ imaging system.

[0299] summary A total of three AAT variants were successfully expressed in ExpiCHO cells at a yield of approximately 300 mg / L (yield estimated after IMAC affinity capture). The N-terminal His tag was fully available for TEV cleavage and was successfully removed for all three variants.

[0300] Example 2 - Effects of wild-type AAT The effect of wtAAT on neutrophil elastase activity was evaluated.

[0301] We administered mist-type wtATT to mice infected with IAV and measured its effect on pneumonia. This resulted in a reduction in lung white blood cell count and lung weight (Figure 2).

[0302] Furthermore, the level of wtAAT in the airways of children with severe pneumonia (such as cystic fibrosis) is approximately 10% higher than that in the blood. 4 It was found to be twice as low. Therefore, severe pulmonary neutrophilic inflammation suppresses the amount of AAT derived from the bloodstream in the lungs. Thus, administering wtAAT by inhalation may provide a higher / appropriate dose depending on the site of the disease / inflammation.

[0303] Example 3 - Design of AAT polypeptide Stability operations for AAT Various amino acid clusters hypothesized to contribute to thermal stability were identified by X-ray crystallography structure determination. These amino acids were found to contribute to thermal stability through improved packing in the hydrophobic core, the addition of favorable interactions across various regions, or an increase in the number of salt bridges. To improve the biophysical properties of wtAAT, each amino acid cluster or mutation was grafted onto wtAAT. All clusters were modeled to ensure that all incoming amino acids fit and that no unfavorable interactions occurred. A total of eight grafts were produced using various numbers of mutant amino acids ranging from 2 to 6 residues (Figures 3a and 3b, Table 3). Information on each graft clustered based on amino acid position is as follows:

[0304] Hydrophobic core: T43S: Introduction of three polar, uncharged side chains to create suitable polar and nonpolar interactions. Present on Helix-A and Helix-B.

[0305] F35: Contrary to its name, phenylalanine 35 was not mutated. Located on β-sheet A facing the hydrophobic core, three residues surrounding residue 35 were mutated. Two of these contribute to a decrease in side chain size, while one contributes to an increase in side chain size. This improves packing of β-sheet A.

[0306] B / C barrel: B / C barrel: Removal of two charged residues on Helix-H facing the B / C barrel, and reduction of the side chain on β-sheet B facing Helix-H. This is to improve hydrophobic packing in this region.

[0307] The addition of six residues between Helix-H and β-sheet B, four of which are charged residues, creates a coordination salt bridge network, stabilizing the native structure. The remaining two residues are involved in increasing (valine to phenylalanine) or decreasing (phenylalanine to alanine) the side chain size. All mutation combinations improve residue packing, providing further stabilization (along with the salt bridge network).

[0308] Citrate binding: Two mutations were introduced into the B / C barrel, one on β-sheet B and the other on β-sheet C. These mutations are located in the region where citrate binds, stabilizing the native state of α1-AT by binding to a surface pocket. By introducing two mutations, both containing larger side chains, this surface pocket is filled, further stabilizing the native state.

[0309] β-sheet C stapling: Adding two charged residues to strands 2 and 3 (s2C and s3C) of β-sheet C creates a salt bridge, which rigidifies and stabilizes β-sheet C.

[0310] Functional area: Breach: Based on the upper surface of β-sheet A and the base of the RCL, five mutations were introduced to increase the existing salt bridge network. The additional salt bridges prevent β-sheet A from rupturing as easily as wtAAT and stabilize its natural conformation.

[0311] Helix-F: Located on the anterior surface of β-sheet A, three mutations were introduced to improve packing between Helix-F and β-sheet A through the addition of one side chain and the reduction of the other two side chains. This hardens the helix and, in some cases, prevents it from easily shifting if β-sheet A opens.

[0312] [Table 9]

[0313] Improvement of AAT thermal stability The purpose of grafting a region onto wtAAT was to increase the thermal stability of the natural state without affecting the AAT function. As a reference point, the natural AAT is T m The temperature is 60°C. To identify the region important for improving thermal stability, circular dichroism fusion tests were performed on each of the eight grafts, including the silent graft (Table 4). Each sample was heated to 25–95°C and then cooled back down to 25°C. Of the six naturally folded grafts, three grafts improved the thermal stability of AAT (Breach and Helix-F grafts). The F35 graft underwent two unfolding transitions: an initial unfolding transition at 65°C and a second transition that continued up to 95°C.

[0314] [Table 10]

[0315] Increased thermal stability correlates with increased thermodynamic stability. 271 The thermodynamic stability of each graft was estimated using thermal deformation curves and van't Hoff analysis and compared to wtAAT. The thermodynamics of each graft are suggestive and provide a rough estimate of how stable each graft is compared to wtAAT. Using thermal deformation curves, Breach and Helix-F are suggested to be more thermodynamically stable than wtAAT (-1.54±0.58 and -4.06±0.8, respectively). Due to the nature of its folding transition, the thermodynamic stability of the F35 graft could not be calculated.

[0316] The increased thermal stability did not significantly impair functionality. To ensure that increased thermal stability did not impair its function, the inhibitory activity of the three most thermally stable grafts (Breach, Helix-F, and F35) was tested against the target protease of AAT, human neutrophil elastase (HNE), by inhibitory stoichiometry (SI) assays. Inhibitory stoichiometry calculates the number of moles of AAT polypeptide required to inhibit one mole of protease. AAT has an inhibitory stoichiometry (SI) of 1:1 against HNE. The inhibitory stoichiometry of the Breach and F35 grafts is identical to that of wtAAT, while Helix-F shows an increased SI (Table 5).

[0317] [Table 11]

[0318] Thermally stable grafts produce more monomeric proteins after refolding. AAT folds through at least one agglutinating intermediate, which also increases the tendency to aggregate during refolding (Kwon et al.). Because it is difficult to calculate and compare the amount of monomeric protein refolded during protein purification (due to inconsistencies in bacterial cell weight), the most accurate way to determine the yield of monomeric AAT after refolding is to start from a known protein concentration. Each protein was unfolded in 5M guanidine hydrochloride (GndHCl) for 2 hours, then refolded by dilution (1:10 dilution) until the final protein concentration was 2 μM. wtAAT aggregated significantly during refolding, resulting in a monomeric protein yield of 11%. The yields of each of the three thermostable grafts were higher than the yield of wtAAT. The F35 graft produced the most monomeric protein, followed by Breach, and finally the Helix-F graft.

[0319] The folding intermediate exists at higher concentrations of the denaturing agent. wtAAT folds through at least one folding intermediate with a tendency to aggregate. Introducing mutations into the peptide may, in some cases, affect the folding intermediate and thus the folding pathway. The exogenous fluorescent dye Bis-ANS binds to the hydrophobic region of the protein, enabling the detection of the folding intermediate. The folding intermediate of AAT is present over a wide range of denaturant concentrations. The three thermostable grafts also have intermediates present over a wide range of concentrations and exhibit fluorescence profiles similar to wtAAT. However, the concentrations at which fluorescence increases differ. For each of the three grafts, the maximum fluorescence intensity appears at a higher denaturant concentration than in wtAAT. Therefore, each graft requires a slightly higher chemical denaturation concentration to promote unfolding.

[0320] Consideration Grafts that increased the thermal stability of α1-AT (AAT) each have mutations in regions that play a role in folding. The F35 graft introduced a mutation in the hydrophobic core, the first region to be folded, while the mutation in the breach region causes misfolding and polymerization during folding (as observed in the Z mutation). During folding, Helix-F is highly disintegrative and undergoes conformational changes during unfolding and polymerization. During the folding pathway of α1-AT, the folding intermediate is prone to aggregation, which can cause aggregation during refolding. The post-refold yield for each graft increased compared to wtAAT, with the F35 graft showing the greatest increase in yield, while the Helix-F graft only slightly increased the yield. This is plausible because the F35 graft is modeled on improved packing and the introduction of favorable interactions, potentially increasing the folding rate and reducing the time the aggregation-prone intermediate is present. Furthermore, the increased yield of the Breach graft may be a result of an increased number of salt bridges that force closure of β-sheet A during the final stage of folding.

[0321] The improved packing modeled in the Helix-F graft did not appear to contribute to the yield after refolding as much as in the other two grafts. This suggests that this graft, and Helix-F in particular, does not contribute to the reduction in aggregation tendency during folding. Since the three highly thermally stable grafts provided desirable interactions within wtAAT, we then used these three grafts to design further composite grafts.

[0322] Example 4 - Further Design of AAT Polypeptides and Their Biophysical Analysis By grafting various amino acid clusters onto AAT, three grafts were produced that exhibited improved thermal stability and increased monomeric protein after refolding, all of which had virtually no effect on the inhibitory function against HNE (only the Helix-F graft had a slightly higher SI). Therefore, it was hypothesized that combining these three grafts would produce an additive thermal stability effect. A complex graft, referred to as "sAAT," was designed, synthesized, and cloned in the same manner as the single graft. The sequence of sAAT is shown in Figure 1.

[0323] Recombinant expression of the sAAT graft resulted in the majority of the protein being in the insoluble fraction (as with all previous grafts), with a small amount remaining soluble. The sAAT graft successfully refolded into its active native conformation.

[0324] The addition of three thermally stable grafts to the sAAT resulted in an additive midpoint in the thermal denaturation of the sAAT, determined by circular dichroic fusion (cooling continuing from 25 to 95°C). The sAAT graft has an unfolding midpoint of 73.4°C. mThis was shown (Figure 4B). Although many attempts have been made to increase the thermal stability of AAT, the sAAT polypeptide has the highest thermal stability of any manipulated native AAT to date (calculated by the circular dichroism thermal denaturation curve). sAAT did not reversibly refold upon cooling from 95°C, but instead formed a precipitate similar to wtAAT (Figure 4A).

[0325] To observe whether further improvements in thermal stability were possible, additional mutations were introduced into sAAT grafts to produce further engineered AAT polypeptides, uAAT. The mutations were as follows:

[0326] [Table 12]

[0327] The amino acid sequence of uAAT is shown in Figure 1.

[0328] Unlike sAAT, which is mainly expressed in the insoluble fraction, uAAT is expressed in a soluble form during recombinant expression.

[0329] Thermodynamic stability was estimated using van't Hoff analysis in the same manner as described above. The thermodynamic stability of the sAAT graft was above WT at 8.66 kcal mol. -1 This was the case (Table 6).

[0330] [Table 13]

[0331] The increased stability of the sAAT polypeptide suggests a potential stability-function trade-off. To ensure that the sAAT polypeptide remains an active inhibitor against HNE, the SI was calculated, and sAAT inhibited HNE with an SI of 1.28 ± 0.1 (Figure 4C and Table 7). This SI value was only slightly higher than that of wtAAT, but not significantly higher.

[0332] Experiments in which uAAT was subjected to thermal denaturation revealed a significant increase in thermal stability compared to wtAAT (Table 7). This is an extremely thermally stable AAT.

[0333] [Table 14]

[0334] Each exemplary AAT polypeptide exhibited an increase in yield after refolding. Under the same conditions, the sAAT graft yielded 63%, supporting the hypothesis (Figure 4D). The folding intermediate remained unaffected and was present over a wide range of concentrations. The greatest difference in the study of the folding intermediate was the denaturant concentration of peak fluorescence compared to wtAAT and each thermally stable graft (peak fluorescence at approximately 2M GndHCl) (Figure 4E). Therefore, higher denaturant concentrations are required to unfold the sAAT polypeptide without affecting the intermediate.

[0335] Enhanced thermal stability increased the yield of monomeric proteins after refolding. The refold yields of the AAT polypeptides described here demonstrate the relationship between thermal stability and refold yield. sAAT and uAAT produced significantly more refolded monomeric proteins than wtAAT. uAAT also followed this relationship. The maximum refold yield was 70%. uAAT produced two more thermally stable monomeric proteins, strongly highlighting the relationship between thermal stability and refold yield.

[0336] [Table 15]

[0337] Example 5 - Anti-inflammatory and immunomodulatory effects of AAT polypeptide Recombinant AATs (sAAT, uAAT, rAAT) were produced according to this disclosure. In vitro experiments in neutrophils stimulated with influenza A virus to produce NETs showed that the AAT polypeptides according to this disclosure reduced NE expression in various stimulation models (Figure 5).

[0338] Further experiments were performed using sAAT. Recombinant AAT (sAAT) was produced by a bacterial expression system. sAAT reduced NE activity using a standardized dose-increasing curve (Figure 6). The manipulated AAT polypeptides described herein were observed to be highly effective in reducing NE activity at various concentrations, both in vitro and in vivo.

[0339] In vitro experiments in neutrophils stimulated to produce NETs showed that sAAT reduces NE expression in various stimulation models (NTHi (infection with Haemophilus influenzae type 1 (NTHi)), IAV (influenza A virus), black carbon (for air pollution), silica (for pulmonary fibrosis), and tobacco smoke extract (CSE)) (Figures 7-10).

[0340] Example 6 - Antiviral effect of AAT polypeptide The recombinant AAT polypeptide described herein was shown to reduce infection in human epithelial cells (Figure 11). Epithelial cells were infected with IAV and rAAT, and intracellular infection was determined using a plaque assay.

[0341] Human lung epithelial cell lines were infected with IAV for 1 hour with and without rAAT (sAAT), then the cells were lysed and the supernatant was cultured for IAV levels using a plaque assay (n=6). sAAT reduced infection of lung epithelial cells (Figure 17).

[0342] Example 7 - AAT polypeptide retains activity after storage. A key factor in distinguishing the recombinant AAT polypeptides described herein from wtAAT is their stability, which is particularly important for nebulization.

[0343] The neutrophil NET assay described above was used again.

[0344] The effects of sAAT stored in a refrigerator for 2 weeks and 5 months, followed by storage at room temperature (RT), were compared with those of fresh wtAAT (referred to as eluted AAT or eAAT) stored at RT. Both sAAT storage samples were useful in reducing NE activity and performed better than fresh wtAAT. The results are shown in Figure 12. The figure shows neutrophils infected with influenza A virus (IAV) that induce increased neutrophil elastase (NE) activity. This was significantly reduced by both sAAT after 2 weeks and 5 months (stored in a refrigerator), and the effect was more pronounced than that of plasma-derived eluted AAT (referred to as wtAAT, eAAT) (stored in a refrigerator for 2 days). This result demonstrates the stability of the AAT polypeptide of this disclosure, which remained potent even after 5 months in a refrigerator.

[0345] Three subtypes of recombinant AAT were produced by the National Biologics Facility at the University of Queensland. This enabled the production of significantly larger quantities of recombinant AAT than bacterial expression systems using Chinese hamster ovary (CHO) expression systems. The three variants had similar effects in reducing NE activity (Figure 13). The efficacy of recombinant AAT variants left at room temperature for one week to reduce NE activity was also evaluated using the NET assay (Figure 14) and standard curves (Figure 15), and their efficacy was demonstrated.

[0346] Recombinant AAT (sAAT) was obtained in mist form, and its effectiveness in reducing NE activity was evaluated using a NET assay (Figure 16). This again demonstrates the stability of the recombinant AAT polypeptide according to this disclosure.

[0347] To evaluate the effect on pneumonia, in vivo experiments were conducted. Mice were infected with IAV. Then, for three days, the mice were either given mist-type rAAT (sAAT) daily or left untreated (control). After this period, the mice were sacrificed. Subsequently, the weight and lung BAL cell count of each mouse were measured. sAAT reduced inflammation by decreasing both lung white blood cell count (Figure 18) and lung weight (Figure 19).

[0348] Mice infected with either seasonal or pathogenic IAV were administered recombinant AAT daily starting the day after infection, followed by more detailed experiments (to mimic clinical practice). Mice were administered AAT in mist form for 5 days and sacrificed on day 4 or day 11 post-infection. In both IAV infections, recombinant AAT had a significant anti-inflammatory effect, accompanied by 1) reduced body weight loss (Figure 20), 2) reduced lung weight (Figure 21), 3) reduced lung white blood cell count (Figure 22), and 4) reduced lung neutrophil elastase (Figure 23). Other preliminary results demonstrated reduced lung cytokine expression (e.g., interleukin-6) and less inflammation in lung histology (including a reduction in areas with pulmonary edema and bronchopneumonia changes). While preliminary results demonstrated a somewhat mild reduction in IAV load, the primary effect is likely the anti-inflammatory effect, as AAT was administered one day after infection.

[0349] Finally, to enhance the usefulness of the present invention, recombinant AAT was freeze-dried and then reconstituted for use in the in vitro NET assay described above. These results demonstrated that freeze-dried AAT remained highly effective in reducing NE activity in vitro (Figure 24).

[0350] summary The results described above demonstrate that exemplary AAT polypeptides reduce inflammatory responses to infections and air pollutants (such as tobacco smoke and air pollution) that cause a variety of common diseases. Furthermore, the AAT polypeptides designed here are highly stable and resistant to aggregation, both of which are advantageous properties for administration via inhalation. Such a route of administration is preferable for enhancing its potent anti-inflammatory effect. The stability of the AAT polypeptides designed here also provides advantages during distribution and storage, enabling the maintenance of their therapeutic effect over extended periods. The high protein stability of the AAT polypeptides disclosed here is also advantageous for certain routes of administration, such as aerosol delivery.

[0351] Those skilled in the art will understand that this disclosure may be subject to variations and modifications other than those specifically described herein. This disclosure will be understood to encompass all such variations and modifications.

[0352] References Levina, V., Dai, W., Knaupp, AS, Kaiserman, D., Pearce, MC, Cabrita, LD, Bird, PI, and Bottomley, SP (2009) Expression, purification and characterization of recombinant Z α1-Antitrypsin-The most common cause of α1-Antitrypsin deficiency. Protein Expr. Purif. 68, 226-232. Grant, SGN, Jesseet, J., Bloomt, FR, and Hanahan, D. (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants 87, 4645-4649. Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. 81, 2035-2039. Studier, F. W., and Moffattf, B. A. (1986) Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes 113-130. Gottesman, S., Halpern, E., and Trisler, P. (1981) Role of sulA and sulB in Filamentation by Lon Mutants of Escherichia coli K-12 148, 265-273. Cabrita, L. D., Dai, W., and Bottomley, S. P. (2006) A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnol. 6, 12. Pearce, M. C., and Cabrita, L. D. (2011) Production of Recombinant Serpins in Escherichia coli, in Methods in Enzymology 1st ed., pp 13-28. Elsevier Inc. Qiagen. (2003) The QIA expressionist Handbook, 5th Ed. Diagen GmbH, Diisseldorf, Ger. Bachman, J. (2013) Site-directed mutagenesis. Methods Enzymol. 1st ed. Elsevier Inc. Horvath, A. J., Lu, B. G. C., Pike, R. N., and Bottomley, S. P. (2011) Methods to measure the kinetics of protease inhibition by serpins. Methods Enzymol. 1st ed. Elsevier Inc. John, D. M., and Weeks, K. M. (2000) Van’t Hoff enthalpies without baselines. Protein Sci. 9, 1416-1419. Greenfield, N. J. (2009) Using circular dichroism collected as a funcion of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat. Protoc. 1, 2527-2535. Matthews, B. W., Nicholson, H., and Becktel, W. J. (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. Acad. Sci. U. S. A. 84, 6663-7. Seo, E. J., Lee, C., and Yu, M. H. (2002) Concerted regulation of inhibitory activity of α1-antitrypsin by the native strain distributed throughout the molecule. J. Biol. Chem. 277, 14216-14220. Kwon, K. S., Lee, S., and Yu, M. H. (1995) Refolding of alpha 1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation. Biochim. Biophys. Acta 1247, 179-84. King PT, Sharma R, O'Sullivan K, Selemidis S, Lim S, Radhakrishna N, Lo C, Prasad J, Callaghan J, McLaughlin P, Farmer M, Steinfort D, Jennings B, Ngui J, Broughton BR, Thomas B, Essilfie AT, Hickey M, Holmes PW, Hansbro P, Bardin PG, Holdsworth SR. Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression. PLoS One 2015; 10: e0120371. King PT, Sharma R, O'Sullivan KM, Callaghan J, Dousha L, Thomas B, Ruwanpura S, Lim S, Farmer MW, Jennings BR, Finsterbusch M, Brooks G, Selemidis S, Anderson GP, ​​Holdsworth SR, Bardin PG. Deoxyribonuclease 1 reduces the pathogenic effects of cigarette smoke exposure in the lung. Sci Rep 2017; 7 : 12128 . Dousha L, Sharma R, Lim S, Ngui J, Buckle AM, King PT. Assessing Respiratory Immune Responses to Haemophilus Influenzae. J Vis Exp King PT, Dousha L, Clarke N, Schaefer J, Carzino R, Sharma R, Wan KL, Anantharajah A, O'Sullivan K, Lu ZX, Holdsworth SR, Ranganathan S, Bardin PG, Armstrong DS. Phagocyte extracellular traps in children with neutrophilic airway inflammation. ERJ Open Res 2021; 7. Janciauskiene S, Wrenger S, Immenschuh S, Olejnicka B, Greulich T, Welte T, Chorostowska-Wynimko J. The Multifaceted Effects of Alpha1-Antitrypsin on Neutrophil Functions. Front Pharmacol 2018; 9: 341. Bai X, Hippensteel J, Leavitt A, Maloney JP, Beckham D, Garcia C, Li Q, Freed BM, Ordway D, Sandhaus RA, Chan ED. Hypothesis: Alpha-1-antitrypsin is a promising treatment option for COVID-19. Med Hypotheses 2021; 146: 110394.

Claims

1. An alpha-1 antitrypsin (AAT) polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, An AAT polypeptide, wherein the amino acid sequence differs from SEQ ID NO: 2 in that it contains three or more amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

2. The above amino acid sequence is one of the following groups of amino acid substitutions from SEQ ID NO: S276K, T278E, T323E, D325N and K327E, L275F, I324V and M358I, and G99A, T144W, and Y171A The AAT polypeptide according to claim 1, having one, two, or all of the above.

3. The AAT polypeptide according to claim 1 or 2, wherein the amino acid sequence has five or more, six or more, or eight or more amino acid substitutions from SEQ ID NO: 2, which is independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

4. The above amino acid sequence has the following amino acid substitutions from SEQ ID NO: An AAT polypeptide according to any one of claims 1 to 3, comprising each of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I.

5. The AAT polypeptide according to any one of claims 1 to 4, wherein the amino acid sequence comprises one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R.

6. The AAT polypeptide according to claim 5, wherein the amino acid sequence comprises two or more, three or more, four or more, five or more, or each of the following further amino acid substitutions from SEQ ID NO: F35L, T43A, T52A, A54G, S365A, and K371R.

7. The AAT polypeptide according to any one of claims 1 to 6, wherein the amino acid sequence has at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with respect to SEQ ID NO:

2.

8. The AAT polypeptide according to any one of claims 1 to 7, wherein the amino acid sequence has 35 or fewer, 30 or fewer, 25 or fewer, and 20 or fewer amino acid modifications from SEQ ID NO:

2.

9. The aforementioned amino acid sequence is It contains three or more amino acid substitutions from Sequence ID No. 2, independently selected from the group consisting of G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, and Depending on the circumstances, it may include one or more further amino acid substitutions from SEQ ID NO: 2, independently selected from the group consisting of F35L, T43A, T52A, A54G, S365A, and K371R. In addition, the AAT polypeptide according to any one of claims 1 to 8, having 10 or fewer amino acid modifications from SEQ ID NO: 2, 5 or fewer amino acid modifications, or having amino acid modifications.

10. The AAT polypeptide according to any one of claims 1 to 9, comprising an additional N-terminal and / or C-terminal amino acid sequence.

11. The AAT polypeptide according to claim 10, comprising an N-terminal sequence MENLYFQGAAS (SEQ ID NO: 7) or MPSSVSWGILLLAGLLCCLVPVSLAEDPQGDAAQKTDTSHH (SEQ ID NO: 8) prior to the amino acid sequence.

12. The AAT polypeptide according to any one of claims 1 to 11, having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

13. The AAT polypeptide according to any one of claims 1 to 12, having improved thermal stability compared to wild-type human AAT.

14. The AAT polypeptide according to any one of claims 1 to 13, having improved refold yield compared to wild-type AAT.

15. The AAT polypeptide according to any one of claims 1 to 14, comprising modifications selected from the group consisting of glycosylation, PEGylation, prenylation, acylation, biotinylation, phosphorylation, and conjugation to a lipid moiety.

16. A nucleic acid molecule encoding the AAT polypeptide according to any one of claims 1 to 15.

17. A vector comprising a nucleic acid sequence encoding the AAT polypeptide according to any one of claims 1 to 15.

18. The vector according to claim 17, wherein the nucleic acid sequence is linked to an expression control sequence suitable for expression in host cells.

19. A cell containing nucleic acid according to claim 16.

20. The cell according to claim 19, comprising the vector according to claim 17 or 18.

21. The cell according to claim 19 or 20, which is a mammalian cell or a bacterial cell.

22. The cell according to claim 21, which is DH5α, BL21(DE3)pLysS, SG13009, or Rosetta Blue DE3 cell.

23. A method for producing an AAT polypeptide according to any one of claims 1 to 15, a. Transfecting a host cell with a nucleic acid sequence encoding the AAT polypeptide described in any one of claims 1 to 15, b. Culturing transfected host cells in cell culture medium and expressing the AAT polypeptide, c. Recovering the AAT polypeptide from the cell culture medium. A method that includes this.

24. AAT polypeptide according to any one of claims 1 to 15, Pharmaceutically acceptable excipients and A pharmaceutical composition containing the following:

25. The pharmaceutical composition according to claim 24, which retains at least 80%, at least 90%, or at least 95% of its initial neutrophil elastase inhibitory activity after storage at 4°C for three months or six months.

26. An AAT polypeptide according to any one of claims 1 to 15 for use as a pharmaceutical agent, or a pharmaceutical composition according to claim 24 or 25.

27. An AAT polypeptide according to any one of claims 1 to 15, or a pharmaceutical composition according to claim 24 or 25, for use in the prevention or treatment of AAT deficiency or a disease or disorder associated with AAT deficiency.

28. An AAT polypeptide according to any one of claims 1 to 15, or a pharmaceutical composition according to claim 24 or 25, for use in the treatment or prevention of inflammatory diseases, respiratory diseases, cancer and / or viral infections.

29. A method for preventing or treating AAT deficiency or a disease or disorder associated with AAT deficiency in a subject, comprising administering to the subject an effective amount of the AAT polypeptide described in any one of claims 1 to 15, or the pharmaceutical composition described in claim 24 or 25.

30. A method for treating or preventing inflammatory diseases, respiratory diseases, cancer and / or viral infections in a subject, comprising administering to the subject an effective amount of the AAT polypeptide described in any one of claims 1 to 15, or the pharmaceutical composition described in claim 24 or 25.

31. Use of an AAT polypeptide according to any one of claims 1 to 15 for the manufacture of a drug for preventing or treating AAT deficiency or a disease or disorder associated with AAT deficiency.

32. Use of the AAT polypeptide according to any one of claims 1 to 15 for the manufacture of agents for treating or preventing inflammatory diseases, respiratory diseases, cancer and / or viral infections.

33. The method, use, or AAT polypeptide or pharmaceutical composition for use according to any one of claims 28, 30, or 32, wherein the inflammatory disease, respiratory disease and / or viral infection is selected from the group consisting of sepsis, autoimmune diseases (e.g., inflammatory arthritis, vasculitis), acute respiratory disease, emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, respiratory disease associated with air pollution, cancer associated with protease activity (e.g., lung cancer), influenza A virus (IAV), and highly pathogenic avian influenza (HPAI).

34. A method, use, or AAT polypeptide or pharmaceutical composition for use according to any one of claims 26 to 33, achieving one or more of the following: reduction of cytokine levels in the target, reduction of extracellular trap formation in the target organ, reduction of protease activity, and reduction of leukocyte infiltrates in the target organ.

35. The method, use, or use of an AAT polypeptide or pharmaceutical composition for use according to any one of claims 26 to 34, wherein the disease or disorder is an inflammatory disease or disorder, and a neutrophil-related inflammatory disease or disorder.

36. The method, use, or use of the AAT polypeptide or pharmaceutical composition according to any one of claims 26 to 35, wherein the AAT polypeptide or pharmaceutical composition is administered in combination with a further therapeutic agent.

Images