Highly fucosylated recombinant human alpha-1 antitrypsin (AAT) protein with immunomodulatory activity and compositions containing same

Abstract

Provided herein is a recombinant AAT protein having a higher fucosylation level and enhanced immunomodulatory biological activity compared to plasma-derived AAT, as well as compositions containing the same and methods for preparing the same.

Description

[Technical Field]

[0001] The present disclosure relates to a recombinant human AAT protein having a high fucosylation level, compositions comprising the same, and methods for preparing the same. Additionally, a method for using the recombinant human AAT protein as an immunomodulator in a subject in need thereof is provided. [Background technology]

[0002] Endogenous human α1-antitrypsin (AAT) is an acute-phase glycoprotein whose abundance in the blood increases approximately fourfold in response to acute inflammation. Hepatocytes are the primary source of AAT, but AAT is also expressed in mononuclear phagocytes and neutrophils, megakaryocytes, pancreatic islet cells, and intestinal epithelial cells. Plasma AAT is a 52 kDa single-chain protein synthesized as a 418-amino acid precursor. Loss of a 24-aa (amino acid) signal peptide generates a 394-amino acid mature protein containing three complex carbohydrate side chains linked by N-glycosidic bonds to three asparagine residues (Asn46, Asn83, and Asn247). As a protease inhibitor, AAT's primary role is to maintain the protease / antiprotease balance in the lung, protecting tissues from inflammatory cell enzymes, particularly human neutrophil elastase (HNE), released by neutrophils during inflammatory conditions. In addition to its high specificity for HNE, AAT also has the ability to neutralize several other proteases, including cathepsin G, proteinase 3, metalloproteases, and cysteine ​​and aspartic acid proteases. Normal plasma concentrations of alpha-1 antitrypsin range up to 3.5 mg / ml. Under certain conditions, AAT diffuses into tissue spaces and forms a 1:1 complex with target proteases. The enzyme / inhibitor complex is then removed from the circulation by binding to a serpin-enzyme complex receptor and degraded by the liver and spleen. [1] AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In patients with AATD, treatment with plasma-derived AAT (pdAAT), known as "augmentation therapy," provides clinical improvement but requires weekly intravenous infusion.

[0003] Postischemic reperfusion (IR) is a pathological condition characterized by the restoration of perfusion and concomitant reoxygenation after an initial restriction of blood supply (oxygen) to an organ.[2] Occlusion of the arterial blood supply results in a severe imbalance of metabolic supply and demand, causing tissue hypoxia. The subsequent restoration of blood flow and reoxygenation is associated with exacerbated tissue injury and a profound inflammatory response.[3] Ischemic injury is associated with systemic inflammation due to cytokine production and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells.[4]

[0004] Many pathological processes contribute to I / R-related tissue injury. Hypoxia is associated with impaired endothelial cell barrier function, which results from a decrease in adenylate cyclase activity and intracellular cAMP levels, and a concomitant increase in vascular permeability and leakage.

[0005] Periods of ischemia are associated with significant changes in the transcriptional regulation of gene expression. For example, ischemia is associated with the inhibition of oxygen-sensing prolyl hydroxylase (PHD) enzymes because these enzymes require oxygen as a cofactor. Hypoxia-associated inhibition of PHD enzymes leads to post-translational activation of hypoxic and inflammatory signaling cascades, which regulate the stability of the transcription factors hypoxia-inducible factor (HIF) and nuclear factor-κB (NF-κB), respectively. Reperfusion injury is characterized by an autoimmune response, which involves natural antibody recognition of neoantigens and subsequent activation of the complement system. Activation of innate and adaptive immune responses also occurs, leading to damage, including activation of pattern recognition receptors such as TLRs and trafficking of inflammatory cells to diseased organs [3].

[0006] Furthermore, ischemia and reperfusion result in the activation of cell death programs, including apoptosis (nuclear fragmentation, plasma membrane blebbing, cell shrinkage, and loss of mitochondrial membrane potential and integrity), autophagy-associated cell death (cytoplasmic vacuolization, loss of organelles, and accumulation of vacuoles with membrane vortices), and necrosis (progressive cell and organelle swelling, plasma membrane rupture, and leakage of proteases and lysosomes into the extracellular compartment) [2,3]. IR-induced tissue injury contributes to morbidity and mortality in a wide range of pathologies, including myocardial infarction (MI), ischemic stroke, acute kidney injury, trauma, and circulatory arrest. Ischemia-reperfusion injury is also a major challenge during organ transplantation and cardiothoracic, vascular, and systemic surgery. [3] Tissue injury and cell death through necrotic, necroptotic, pyroptotic, apoptotic, and autophagy mechanisms initially occur as a result of ischemic injury, followed by subsequent damage induced by reperfusion [5]. Furthermore, exposure to ischemia and reperfusion in one organ can subsequently trigger inflammatory activation in other organs, ultimately resulting in multiple organ failure [3]. Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide [6]. After AMI, ischemic damage and the reperfusion injury associated with the restoration of blood flow promote cell death, trigger an inflammatory response, and ultimately induce cardiac dysfunction leading to heart failure [7]. Preclinical and clinical evidence suggests that plasma-derived AAT has several beneficial effects in the treatment of acute MI, possessing cytoprotective and anti-inflammatory properties [8, 9].

[0007] Intestinal ischemia is a common (>1 in 1,000 hospital admissions) and life-threatening condition. In-hospital mortality ranges from 67% to 80%, primarily due to: 1) delayed diagnosis due to the lack of specific biomarkers; 2) the major risk of sepsis; and 3) the lack of effective treatment. In fact, intestinal ischemia is often diagnosed when the intestine is in a necrotic state, requiring resection and compromising survival

[10] . Reperfusion triggers massive ROS formation, which activates innate immune molecular (chemokine / cytokine / coagulation cascade) and cellular (neutrophil / macrophage / lymphocyte / platelet) components, resulting in local and systemic inflammation and cell death. As a result, ischemia-reperfusion injury (IRI) can disrupt the mucosal barrier, allowing bacterial (endotoxin) translocation and sepsis

[11] .

[0008] Studies in animals and humans have shown that prolonged ischemia followed by reperfusion compromises the paracellular barrier of the epithelial lining, allowing easy translocation of bacterial products such as endotoxins.

[12] Endotoxins such as lipopolysaccharide (LPS) bind to Toll-like receptor-4 (TLR-4), activating various immune responses and leading to the release of proinflammatory cytokines.

[13]

[0009] A tightly junctional monolayer of epithelial cells forms a barrier between the intestinal lumen and the innate immune cells in the underlying lamina propria. This epithelial lining is constantly exposed to environmental factors and bacteria. In recent years, intestinal epithelial cells have been increasingly recognized as important mediators of inflammation

[14] . Thereby, the epithelium actively contributes to antimicrobial host defense and the maintenance of mucosal homeostasis.

[0010] Key pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and TNFα, are potentiated by serine proteases and are therefore blocked by serine protease inhibitors, particularly AAT

[15] . Furthermore, AAT induces the production and release of anti-inflammatory mediators, such as IL-10 and IL-1 receptor antagonist (IL-IRa)

[16] .

[0011] Glycosylation plays an important role in determining the immunomodulatory properties of AAT. It has been shown that changes in AAT glycans during inflammation and malignant conditions can affect protein function. Fucosylation is a type of glycosylation that is often considered non-informative and is therefore removed or otherwise used only as a biomarker.

[0012] A recombinant form of human AAT has been described and is intended for use in the treatment of AAT-deficient emphysema [6]. For example, U.S. Patent No. 8,357,661 relates to recombinant human α-1-antitrypsin. For example, Marie-Eve Lalonde et al. describe the production of α2,6-sialylated and nonfucosylated recombinant α-1-antitrypsin in CHO cells. For example, Izel Koyuturk et al. disclose the high-level production of wild-type and oxidation-resistant recombinant α-1-antitrypsin in glycoengineered CHO cells. For example, Mary Ann Comunale et al. describe the combination of specific fucosylation of α-1-antitrypsin in patients with liver cirrhosis and cancer: its potential as a biomarker for hepatocellular carcinoma (HCC). For example, Ogawa K. et al. reported on the triantennary trisialylated monofucosylated glycan of α-1-antitrypsin as a novel glycobiomarker for nonalcoholic steatohepatitis. Cormac McCarthy et al. reported on the role and importance of acute phase protein glycosylation, focusing on α-1-antitrypsin in acute and chronic inflammatory conditions. Cormac McCarthy et al. reported on the increase in outer arm and core fucose residues on the N-glycans of mutant α-1-antitrypsin proteins from α-1-antitrypsin-deficient individuals. Xiaojuan Zhang et al. reported on α-1-antitrypsin as a novel biomarker and potential therapeutic approach for metabolic diseases.

[0013] Thus, there is a need in the art for improved recombinant human AAT compositions that are safe, efficient, and cost-effective to produce, and that have enhanced beneficial biological activities and minimized side effects compared to plasma-derived AAT. Summary of the Invention

[0014] According to some embodiments, the present invention provides recombinant human α1-antitrypsin (rAAT), which shows higher fucosylation level in target tissues and enhanced immunomodulatory effect compared with plasma-derived AAT (pdAAT).Furthermore, the present invention provides a composition comprising advantageous recombinant AAT, its preparation and purification method from producing cells, and its use method for inducing immunomodulatory effect and treating various inflammation-related symptoms.Such immunomodulatory effect includes but is not limited to the effects of reducing inflammation (anti-inflammatory activity), increasing anti-inflammatory cytokines, reducing cell damage, tissue protection, wound healing, etc., or any combination thereof.

[0015] According to some embodiments, the advantageous rAAT disclosed herein comprises the full-length amino acid sequence of the human wild-type (WT) protein and surprisingly exhibits highly improved immunomodulatory effects compared to the corresponding plasma-derived (i.e., native protein) human AAT having the same amino acid sequence.

[0016] According to some embodiments, the advantageous rAAT disclosed herein, produced in Chinese hamster ovary (CHO) cells, exhibits very high levels of fucosylated N-linked glycans, and even more surprisingly, such high levels of fucosylation (at least 75% or more) can achieve enhanced biological activity of the rAAT as an immunomodulatory agent both in vitro and in vivo, as shown herein below.

[0017] Thus, according to some embodiments, the rAAT disclosed herein surprisingly demonstrates activity as a neutrophil elastase inhibitor, with its immunomodulatory activity exceeding that of plasma-derived AAT. Interestingly, as detailed herein, the final clone selected after screening for immunomodulatory efficacy exhibits a significantly higher level of fucosylation on N-linked glycans compared to other less efficient clones.

[0018] According to some embodiments, and without wishing to be bound by any theory or mechanism, surprisingly, the high level of fucosylation of recombinant AAT is at least partially responsible for the improved and enhanced immunomodulatory and anti-inflammatory effects (including tissue protective effects) of recombinant AAT compared to plasma-derived AAT protein that exhibits only low levels of fucosylation (approximately 10-fold fewer fucosylated N-glycans).

[0019] Thus, according to some embodiments, there is provided recombinant human alphal-antitrypsin (rhAAT) comprising at least about 80% fucosylated N-linked glycans.

[0020] According to some embodiments, the rhAAT comprises at least about 85%, at least about 90%, at least about 93% fucosylated glycans.

[0021] According to some embodiments, greater than about 70%, greater than about 80%, greater than about 85%, or greater than about 90% of the fucose units are core fucose.

[0022] According to some embodiments, about 60-80% of the fucosylated N-linked glycans are diantennary. According to some embodiments, about 70% of the fucosylated N-linked glycans are diantennary.

[0023] According to some embodiments, about 5-20% of the fucosylated N-linked glycans are triantennary. According to some embodiments, about 10% of the fucosylated N-linked glycans are triantennary.

[0024] According to some embodiments, about 5-20% of the fucosylated N-linked glycans are tetraantennary. According to some embodiments, about 10% of the fucosylated N-linked glycans are tetraantennary.

[0025] According to some embodiments, rhAAT exhibits enhanced immunomodulatory activity compared to the activity of purified plasma-derived AAT (pdAAT).

[0026] According to some embodiments, the rhAAT exhibits immunomodulatory activity that is at least about 5% or more, at least about 10% or more, at least about 20% or more, or at least about 50% or more relative to the activity of purified plasma-derived AAT.

[0027] According to some embodiments, the immunomodulatory activity may be characterized by a decrease in the activity, expression and / or secretion levels of one or more of IL-1β, TNFα, IL-6, IL-8, IL-18 and MCP1 / CCL2.

[0028] According to some embodiments, immunomodulatory activity may be characterized by increased activity, expression and / or secretion levels of IL-10 and / or IL-1-receptor antagonist (IL-IRa).

[0029] According to some embodiments, rhAAT can be produced or expressed in a Chinese hamster ovary (CHO) cell line. In some embodiments, the CHO cell line is a CHO DG44 cell line. In some embodiments, the CHO cell line can further express α-2,6-sialyltransferase. In some embodiments, the α-2,6-sialyltransferase is exogenously expressed in the cell.

[0030] According to some embodiments, a pharmaceutical composition is provided comprising rhAAT and a pharmaceutically acceptable carrier.

[0031] According to some embodiments, the pharmaceutical composition is for use in treating or preventing an inflammatory condition in a subject in need thereof.

[0032] According to some embodiments, there is provided a method for preventing or treating an inflammatory condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of rhAAT.

[0033] According to some embodiments, the inflammatory condition is a pulmonary disease selected from the group consisting of alpha 1-antitrypsin deficiency (AATD), small airway disease, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, pneumonia, parenchymal and fibrotic lung diseases or disorders, interstitial pulmonary fibrosis, reinflammation, acute respiratory distress syndrome (ARDS), and sarcoidosis.

[0034] According to some embodiments, the inflammatory condition may be selected from graft-versus-host disease (GVHD), ischemia-reperfusion injury, post-transplant ischemia / reperfusion injury, acute myocardial infarction, acute kidney injury, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease, Crohn's disease, ulcerative colitis, psoriasis, type I diabetes, dermatitis, pneumonia, sepsis, wound healing, and systemic lupus erythematosus. Each possibility is a separate embodiment. According to some embodiments, the inflammatory condition is ischemia-reperfusion injury.

[0035] According to some embodiments, rhAAT or a pharmaceutical composition may be administered at a dose of about 1 mg / kg to about 500 mg / kg. In some embodiments, when rhAAT or a pharmaceutical composition is administered by injection, it may be administered at a dose of about 200 mg / kg to about 350 mg / kg. In some embodiments, when rhAAT or a pharmaceutical composition is administered by inhalation, it may be administered at a dose of about 20 mg / dose to 200 mg / dose.

[0036] According to some embodiments, administration may be performed once per day, 1-3 times per day, 1-7 times per week, 1-4 times per month, or any combination thereof.

[0037] According to some embodiments, the rhAAT or pharmaceutical composition may be administered by injection.

[0038] According to some embodiments, the rhAAT or pharmaceutical composition may be administered by inhalation.

[0039] According to some embodiments, a method for producing rhAAT with a high fucosylation level is provided, comprising diluting and filtering a cell harvest, performing a first step of chromatography (e.g., using a POROS XQ column), performing conductivity adjustment (e.g., by dilution and concentration by ultrafiltration (UF)), performing a second chromatography (e.g., using a Capto Adhere Column), and performing a buffer exchange step (e.g., by ultrafiltration / diafiltration) to obtain purified rAAT protein. The method may further optionally comprise bulk loading and storage under appropriate conditions (e.g., storage at -80°C).

[0040] Further embodiments, features, advantages, and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given below. It should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, is given by way of example only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows a diagram of the plasmid map of the rhAAT expression vector: CMV cytomegalovirus promoter, DHFR-dihydrofolate reductase. [Figure 2] FIG. 2 shows a flow chart of an rAAT purification process according to some embodiments. [Figure 3A] Figures 3A-D show representative results of purification from a lab-scale production run: Figure 3A shows the POROS XQ chromatogram; Figure 3C shows the Capto™ Adhere chromatogram. Figures 3B and 3D show the correlation pictograms of the SDS-PAGE analysis, respectively. rAAT peaks identified in the chromatograms are marked by frames, and the corresponding lanes on the SDS-PAGE gel are marked by arrows. M: Marker; S / St: Standard; H: Harvest; L: Load; U: Unbound; W: Wash; E: Elute; R: Regeneration; F: Flow-through; [Figure 3B] Same as above [Figure 3C] Same as above [Figure 3D] Same as above [Figure 4] FIG. 4 shows a representative chromatogram of the final fraction with the monomer peak area percent shown in the inset table. [Figure 5A] Figure 5A is a bar graph showing the effect of various concentrations of recombinant AAT clones (rhAAT clone 26 and clone 36) and plasma-derived AAT (pdAAT) on LPS-induced KC secretion in splenocytes. Cells were treated with different concentrations of AAT, vehicle, and dexamethasone. KC secretion was determined by ELISA and expressed as a percentage reduction compared to vehicle control. Inhibition of cytokine secretion by rAAT was calculated by comparing cytokine concentrations in the presence or absence of AAT (control). Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are presented for each AAT treatment compared to cells treated with vehicle + LPS according to t-test. [Figure 5B]Figure 5B is a bar graph showing the effect of recombinant AAT clones (rhAAT clone 26 and clone 36) and pdAAT, at various concentrations, on LPS-induced TNF secretion in splenocytes. Cells were treated with different concentrations of AAT, vehicle, and dexamethasone. TNFα secretion was determined by ELISA and expressed as a percentage reduction compared to vehicle control. Inhibition of cytokine secretion by rAAT was calculated by comparing cytokine concentrations in the presence or absence of AAT (control). Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each AAT treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 6A] Figure 6A is a bar graph showing the effect of recombinant AAT and pdAAT, respectively, at various concentrations, on LPS-induced TNF secretion in mouse macrophages. TNFα secretion was determined by ELISA and shown as the percentage reduction compared to vehicle control. Inhibition of cytokine secretion by rAAT was calculated by comparing cytokine concentrations in the presence or absence (control) of AAT. Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001. Statistics are shown for each AAT treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 6B] Figure 6B is a bar graph showing the effect of recombinant AAT versus dAAT at various concentrations on LPS-induced CXCL1 secretion in mouse macrophages. CXCL1 secretion was detected by ELISA and expressed as a percentage reduction compared to vehicle control. Inhibition of cytokine secretion by rAAT was calculated by comparing cytokine concentrations in the presence or absence (control) of AAT. Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each AAT treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 6C] Figure 6C is a bar graph showing the effect of recombinant AAT and pdAAT at various concentrations on LPS-induced IL-10 secretion in mouse macrophages. IL-10 secretion was detected by ELISA and shown as a percentage increase compared to the vehicle control. IL-10 secretion was detected by ELISA and shown as a percentage increase compared to the vehicle control. Results show the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 7A] Figure 7A is a bar graph showing the effect of two recombinant AAT clones (C26 and C36) and pdAAT at various concentrations on LPS-induced IL-6 secretion in PBMC cells. Freshly isolated human PBMCs were treated with different concentrations of AAT, vehicle, and dexamethasone. IL-6 secretion was detected by ELISA and expressed as a percentage reduction compared to vehicle control. Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 7B] Figure 7B is a bar graph showing the effect of two recombinant AAT clones and pdAAT at various concentrations on LPS-induced TNF secretion in PBMC cells. Freshly isolated human PBMCs were treated with different concentrations of AAT, vehicle, and dexamethasone. TNFα secretion was determined by ELISA and expressed as a percentage reduction compared to vehicle control. Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 7C]Figure 7C is a bar graph showing the effect of two recombinant AAT clones and pdAAT at various concentrations on LPS-induced IL-10 secretion in PBMC cells. Freshly isolated human PBMCs were treated with different concentrations of AAT, vehicle, and dexamethasone. IL-10 secretion was detected by ELISA and expressed as a percentage reduction compared to vehicle control. Results represent the mean ± SEM of wells in each group. *p<0.05, **p<0.01, ***p<0.001 statistics are shown for each treatment compared to vehicle + LPS-treated cells according to t-test. [Figure 8A] Figures 8A–C are bar graphs showing the effect of rAAT on left ventricular (LV) contractile function in mice after myocardial ischemia-reperfusion (IR) injury. CD1 mice (8–10 weeks old) underwent 30 minutes of transient left coronary artery occlusion to induce IR, and rAAT or control was intraperitoneally injected immediately after the onset of reperfusion. A sham control was performed without occlusion of the artery. 24 hours after reperfusion, mice were anesthetized with isoflurane (1.5–3%) and echocardiography was performed to measure left ventricular fractional shortening (LVFS) to calculate left ventricular ejection fraction (LVEF) (Figure 8A). Infarct size under different conditions is shown in Figure 8B (% of area at risk) and Figure 8C (infarcted LV segment). Triphenyltetrazolium chloride (TTC) was used to measure IR size. Hearts were excised and perfused with normal saline containing 2.5 mM CaCl2. Subsequently, 1% Evans Blue dye was injected into the aorta, and the heart was then perfused again with saline to wash out excess Evans Blue. Finally, the heart was frozen, cut into sections, and then incubated in 1% TTC isotonic phosphate buffer at room temperature for 30 minutes. The area of ​​infarcted tissue, area at risk, and the entire LV were determined using ImageJ software. Results are expressed as mean ± SEM. [Figure 8B] Same as above [Figure 8C] Same as above [Figure 9A]Figures 9A-H show the effect of rAAT on wound healing in tissue culture cells. The tissue protective activity of rAAT was evaluated in wound healing (gap repair by cell migration) in A549 cells or human umbilical vein endothelial cells (HUVECs). A gap was created in a confluent cell layer by a straight scratch at a 30-degree angle, simulating a wound. Cells were treated with 1 mg / ml pdAAT and two clones of rAAT in 2% serum medium. Controls were treated with vehicle (saline). Figures 9A-F show pictograms at T0 and 24 hours after treatment (T24). Figures 9A-9B show control treatment (saline); Figures 9C-9D show rAAT (clone 26); Figures 9E-9F show rAAT (clone 36); Figure 9G shows a bar graph of the percentage of wound closure on A549 cells calculated by ImageJ software 16 and 24 hours after treatment with control, pdAAT, rAAT (clone 26), and rAAT (clone 36). Results are shown as mean ± SEM. [Figure 9B] Same as above [Figure 9C] Same as above [Figure 9D] Same as above [Figure 9E] Same as above [Figure 9F] Same as above [Figure 9G] Same as above [Figure 9H] Figure 9H shows the effect of rAAT on wound healing in HUVECs. Gaps were created using Ibidi culture inserts. Cells were treated with 1 mg / ml pdAAT, rAAT clone 26, or saline (control) in 5% serum medium. Figure 9H shows a bar graph of the percentage of wound closure calculated by ImageJ software 13 and 18 hours after treatment with control, pdAAT, and rAAT (clone 26). Results are shown as mean ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0042] According to some embodiments, the present invention provides recombinant human AAT that has full-length wild-type sequence and has high fucosylation level (at least more than 75%), which can be produced in large quantities in CHO cell.It is illustrated herein that this recombinant AAT has the same elastase inhibitory activity as pdAAT, and also shows highly excellent immunoregulatory activity.

[0043] According to some embodiments, native plasma-derived AAT contains three glycosylation sites, however, as exemplified herein, it is surprisingly shown that at least 75%, at least 80%, at least 85%, or at least 90% of the N-linked glycans of the recombinant human AAT disclosed herein are fucosylated.

[0044] According to some embodiments, as exemplified herein, the immunomodulatory activity of recombinant AAT was evaluated in cell-based assays using primary cultures from C57BL / 6 male mouse spleens and human peripheral blood mononuclear cells (PBMCs), as well as an in vivo acute myocardial infarction (IMI) model.

[0045] To facilitate understanding of the present invention, several terms and phrases are defined below. It is to be understood that these terms and phrases are for purposes of description and not of limitation, and that a term or phrase herein should be interpreted by one of ordinary skill in the art in light of the teaching and guidance presented herein, in combination with the knowledge of such an artisan.

[0046] As used herein, the terms "AAT" and "AAT-1" can be used interchangeably. These terms refer to human alpha (α) 1 antitrypsin. In some embodiments, AAT has the nucleotide sequence shown by SEQ ID NO: 1 and the amino acid sequence shown by SEQ ID NO: 2.

[0047] The terms "plasma-derived AAT" and "pdAAT" are used interchangeably and refer to AAT derived / purified / produced from human plasma. In some embodiments, pdAAT is a naturally occurring protein.

[0048] The terms "recombinant AAT," "human recombinant AAT," "rAAT," "hrAAT," and "rhAAT" are interchangeable and refer to recombinant AAT expressed from an exogenous expression plasmid introduced into a production cell. A schematic diagram of an exemplary plasmid for expressing recombinant AAT, according to some embodiments, is shown in FIG. 1. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the CHO cell is DG44. In some embodiments, the CHO cell optionally further expresses -2,6 sialyltransferase.

[0049] As used herein, the term "treating" includes, but is not limited to, one or more of the following: abrogating, ameliorating, inhibiting, attenuating, blocking, suppressing, reducing, delaying, arresting, alleviating, or preventing symptoms associated with the condition. Each possibility represents a separate embodiment. In some embodiments, the condition is an immune-related condition, such as an inflammatory condition.

[0050] As used herein, an N-linked glycan is a sugar chain that is covalently attached to an asparagine residue of a polypeptide. N-linked glycans can be branched, resulting in two (referred to herein as "Di" or "di"), three (referred to herein as "Tri" or "tri"), or four (referred to herein as "Tetra" or "tetra") antennae.

[0051] Fucosylation is a type of glycosylation that involves the addition of fucose sugar units to polysaccharides. Fucose is a hexose deoxysugar lacking a hydroxyl group at carbon 6 (C-6) and having an L-configuration. In fucosylated N-linked glycans (i.e., fucose-containing glycan structures), fucose can exist as structure-specific fucosylation—"core fucosylation" or "antennary fucosylation."

[0052] Natural AAT protein has three glycosylation sites.Therefore, AAT contains three N-linked glycans that can be branched.Surprisingly, it has been shown herein that the recombinant AAT disclosed herein has high fucosylation, particularly core fucosylation, in which fucose is linked to the innermost N-glycan residue.

[0053] Thus, according to some embodiments, a recombinant human AAT protein is provided having a degree of fucosylation in the range of about 70-95% or any subrange thereof. In some embodiments, the degree / level of fucosylation is about 80%. In some embodiments, the degree / level of fucosylation is about 85%. In some embodiments, the degree / level of fucosylation is about 90%. In some embodiments, the degree / level of fucosylation is about 93%. In some embodiments, the degree / level of fucosylation is at least about 75%. In some embodiments, the degree / level of fucosylation is at least about 80%. In some embodiments, the degree / level of fucosylation is at least about 85%. In some embodiments, the degree / level of fucosylation is at least about 88%. In some embodiments, the degree / level of fucosylation is at least about 90%. In some embodiments, the degree / level of fucosylation is at least about 91%. In some embodiments, the degree / level of fucosylation is at least about 92%. In some embodiments, the degree / level of fucosylation is at least about 93%. In some embodiments, the degree of fucosylation is greater than about 75%, greater than about 80%, greater than about 85%, or greater than about 90%.

[0054] According to some embodiments, about 50-99% of the fucose units are core fucose units. In some embodiments, more than about 50% of the fucose units are core units. In some embodiments, more than about 60% of the fucose units are core units. In some embodiments, more than about 70% of the fucose units are core units. In some embodiments, more than about 80% of the fucose units are core units. In some embodiments, more than about 85% of the fucose units are core units. In some embodiments, more than about 90% of the fucose units are core units.

[0055] According to some embodiments, about 55-80% of the fucosylated N-linked glycans are diantennary. In some embodiments, at least about 55% of the fucosylated N-linked glycans are diantennary. In some embodiments, at least about 60% of the fucosylated N-linked glycans are diantennary. In some embodiments, at least about 65% of the fucosylated N-linked glycans are diantennary. In some embodiments, at least about 70% of the fucosylated N-linked glycans are diantennary. In some embodiments, at least about 75% of the fucosylated N-linked glycans are diantennary.

[0056] According to some embodiments, about 5-25% of the fucosylated N-linked glycans are triantennary. According to some embodiments, at least about 5% of the fucosylated N-linked glycans are triantennary. According to some embodiments, at least about 7.5% of the fucosylated N-linked glycans are triantennary. According to some embodiments, at least about 10% of the fucosylated N-linked glycans are triantennary. According to some embodiments, at least about 15% of the fucosylated N-linked glycans are triantennary. According to some embodiments, at least about 20% of the fucosylated N-linked glycans are triantennary.

[0057] According to some embodiments, about 5-20% of the fucosylated N-linked glycans are tetraantennary. According to some embodiments, at least about 5% of the fucosylated N-linked glycans are tetraantennary. According to some embodiments, at least about 7.5% of the fucosylated N-linked glycans are tetraantennary. According to some embodiments, at least about 8.5% of the fucosylated N-linked glycans are tetraantennary. According to some embodiments, at least about 10% of the fucosylated N-linked glycans are tetraantennary.

[0058] According to some embodiments, the fucosylation level of rAAT is at least 2-12 fold higher than the fucosylation level of plasma-derived AAT (native AAT). In some embodiments, the fucosylation level of rAAT is at least 2 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 3 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 4 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 5 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 6 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 7 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 8 fold higher than the fucosylation level of pdATT. In some embodiments, the fucosylation level of rAAT is at least 9 times higher than that of pdATT. In some embodiments, the fucosylation level of rAAT is at least 10 times higher than that of pdATT. In some embodiments, the fucosylation level of rAAT is at least 11 times higher than that of pdATT. In some embodiments, the fucosylation level of rAAT is at least 12 times higher than that of pdATT.

[0059] According to some embodiments, various analytical methods can be used to determine the N-glycosylation level, particularly the fucosylation level, of a protein, including, for example, liquid chromatography-mass spectrometry (LC-MS analysis). LC-MS analysis of AAT proteins (recombinant and plasma-derived) is exemplified below (Example 4).

[0060] According to some embodiments, the rAAT disclosed herein can be produced in CHO cells. In some embodiments, the CHO cells are a CHO DG44 cell line. Such cell lines can be transformed (e.g., by transfection) with a plasmid / expression vector (such as the expression plasmid illustrated in FIG. 1 ) containing a nucleotide sequence encoding the open reading frame (ORF) of human α1-antitrypsin (represented by SEQ ID NO: 1) under the control of a promoter, under appropriate conditions, such as an appropriate medium (e.g., serum-free medium). Selectable markers, such as DHFR and neomycin, can be used to identify AAT-expressing clones. In some embodiments, the cells can be further engineered to express α-2,6 sialyltransferase, for example, by transfection of an α-2,6 sialyltransferase coding sequence. In some embodiments, AAT and α-2,6 sialyltransferase can be transfected into CHO cells in separate or common expression vectors. In some embodiments, each gene (i.e., AAT and α-2,6 sialyltransferase) can be under the control of separate or similar promoters.

[0061] Thus, according to some embodiments, a process for the production of rAAT in CHO cells is provided, which process comprises culturing suitable CHO host cells under conditions that allow expression of the rAAT polypeptide from a vector introduced into the cells, and optionally recovering / isolating the produced polypeptide from the cell culture.

[0062] According to some embodiments, a method for recovering rAAT from CHO cells while maintaining the integrity and activity of the protein is provided. Referring to FIG. 2, FIG. 2 schematically illustrates a method for recovering rAAT from CHO cells, which includes, in step 22, obtaining a clarified CHO cell harvest expressing rAAT. In step 24, the harvest is diluted and filtered. The dilution can be any ratio, for example, 1:3 dilution with an appropriate buffer, and the filtration can include, for example, 0.2 μm filtration. In step 26, the sample from step 24 is processed by chromatography, for example, using a POROS XQ column. Chromatographic separation can be repeated for any number of cycles, for example, 1 to 3 cycles. In step 28, the conductivity of the sample is adjusted by dilution with an appropriate buffer and further concentrated by utilizing an ultrafiltration (UF) process. In step 30, the sample is reprocessed by chromatography, for example, using a Capto Adhere column utilized in flow-through mode. In step 32, buffer exchange of the sample is performed using, for example, an ultrafiltration / diafiltration system with a 10 kDa and 0.2 μm cutoff. Then, in a final step 34, the sample is transferred to a suitable container (vial, vessel, etc.) for use or for storage (e.g., storage at 4° C., −20° C., or −80° C.).

[0063] According to some embodiments, the methods disclosed herein have identified clones of rATT with high fucosylation levels and enhanced immunomodulatory activity, also referred to herein as "C-26" and "C-36."

[0064] According to some embodiments, the rAAT obtained from cells can be used as is, or can be used in pharmaceutical compositions in the presence of one or more suitable excipients.Suitable excipients can be selected according to the purpose, type and / or use of the composition.In some embodiments, the excipient is a pharmaceutical excipient, which can include a pharmaceutical carrier, vehicle, buffer and / or diluent.

[0065] According to some embodiments, the rAAT of the compositions disclosed herein may be used as an immunomodulatory agent and as a pharmaceutical for treating various immune-related conditions.

[0066] According to some embodiments, the immunomodulatory activity of rAAT may be characterized by a reduction in the activity, expression and / or secretion levels of one or more of IL-1β, TNFα, IL-6, IL8, IL 18, MCP1 / CCL2.

[0067] According to some embodiments, the immunomodulatory activity may be characterized by increasing the activity or expression levels of IL-10 and IL-1 receptor antagonist (IL-IRa).

[0068] According to some embodiments, as exemplified herein, surprisingly, rAAT exhibits enhanced (increased) immunomodulatory activity compared to plasma-derived AAT under similar conditions, despite having the same amino acid sequence.

[0069] In some embodiments, rAAT exhibits increased immunomodulatory activity compared to plasma-derived (native) AAT (pdAAT). In some embodiments, the increased immunomodulatory activity is about 2-2500% greater. In some embodiments, rAAT exhibits at least about 2% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 5% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 10% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 20% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 30% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 40% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 50% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 60% greater immunomodulatory activity compared to pdAAT. In some embodiments, rAAT exhibits at least about 70% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 80% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 90% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 100% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 150% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 200% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 300% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 400% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 500% greater immunomodulatory activity than pdAAT. In some embodiments, rAAT exhibits at least about 1000% greater immunomodulatory activity than pdAAT.In some embodiments, the rAAT exhibits at least about 2000% greater immunomodulatory activity compared to pdAAT. In some embodiments, the rAAT exhibits at least about 2500% greater immunomodulatory activity compared to pdAAT. In some embodiments, the increased immunomodulatory activity is about 1.5-50 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 5-40 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 10-30 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 16-20 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 1.5 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 2 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 3 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 4 fold greater than the activity of pdAAT. In some embodiments, the increased immunomodulatory activity is about 5-fold greater than the activity of pdAAT.

[0070] According to some embodiments, rAAT or a composition comprising it can be used to treat inflammatory conditions in a subject in need thereof. In some embodiments, the inflammatory condition is a pulmonary disease or condition. In some embodiments, the pulmonary disease or condition can be selected from, but not limited to, alpha-1 antitrypsin deficiency (AATD), small airway disease, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, pneumonia, parenchymal and fibrotic lung disease or disorder, interstitial pulmonary fibrosis, reinflammation, acute respiratory distress syndrome (ARDS) and sarcoidosis. Each possibility is a separate embodiment.

[0071] In some embodiments, the inflammatory condition may be selected from, but is not limited to, graft-versus-host disease (GVHD), ischemia-reperfusion injury, post-transplant ischemia / reperfusion injury, myocardial infarction, acute kidney injury (AKI), rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease, Crohn's disease, ulcerative colitis, psoriasis, type I diabetes, dermatitis, pneumonia, sepsis, wound healing, systemic lupus erythematosus, and multiple sclerosis. Each possibility is a separate embodiment.

[0072] According to some embodiments, rAAT or a composition comprising it may be administered by any suitable route of administration, which may be determined according to the condition to be treated, the type of tissue, the characteristics of the subject, and the like.

[0073] According to some embodiments, the route of administration may include local and systemic routes. Exemplary suitable routes of administration include, but are not limited to, oral, intranasal, parenteral, intravenous, topical, enema, or inhalation. According to another embodiment, systemic administration is via injection. For administration by injection, the composition may be formulated in an aqueous solution, such as a physiologically compatible buffer (e.g., but not limited to, Hank's solution, Ringer's solution, or physiological salt buffer). The formulation for injection may be provided in unit dosage form, for example, in ampoules or in multi-dose containers, optionally containing a preservative.

[0074] According to another embodiment, systemic administration is via a parenteral route. According to some embodiments, parenteral administration is intravenous, intraarterial, intramuscular, intraperitoneal, intradermal, intravitreal, or subcutaneous administration. Each of the above administration routes represents a separate embodiment of the present invention. According to another embodiment, parenteral administration is by bolus injection. According to another embodiment, parenteral administration is by continuous infusion. According to some embodiments, preparations of the compositions of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions, each of which represents a separate embodiment of the present invention. Non-limiting examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

[0075] In another embodiment, parenteral administration is transmucosal administration. In another embodiment, transmucosal administration is nasal administration. For mucosal administration, a penetrant appropriate to the barrier to be permeated is used in the composition. Such penetrants are generally known in the art. The preferred mode of administration will depend on the particular indication being treated and will be apparent to those skilled in the art.

[0076] Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0077] According to another embodiment, the compositions formulated for injection may be in the form of a solution, suspension, dispersion or emulsion in an oily or aqueous vehicle and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Non-limiting examples of suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides.

[0078] According to some embodiments, rAAT or composition is administered intravenously, and therefore is formulated in a form suitable for intravenous administration. According to another embodiment, the composition is administered intraarterially, and therefore is formulated in a form suitable for intraarterial administration. According to another embodiment, the composition is administered intramuscularly, and therefore is formulated in a form suitable for intramuscular administration.

[0079] According to another embodiment, the systemic administration is via the enteral route. According to another embodiment, the administration by the enteral route is buccal administration. According to another embodiment, the administration by the enteral route is oral administration. According to some embodiments, the composition is formulated for oral administration.

[0080] According to some embodiments, oral administration is in the form of hard or soft gelatin capsules, pills, capsules, coated tablets, tablets including dragees, elixirs, suspensions, liquids, gels, slurries, syrups or inhalations, and controlled release forms thereof.

[0081] According to some embodiments, administration can include any suitable dosing regimen, depending on, among other things, the medical condition, patient characteristics, route of administration, etc. In some embodiments, administration can include a single administration. In some embodiments, administration can include once daily, twice daily, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, every 2 weeks, every 3 weeks, monthly, etc. In some embodiments, administration can include a loading dose followed by maintenance administration at increasing intervals, e.g., a loading dose on day 0, maintenance doses on days 1, 3, 5, and 7, followed by weekly or biweekly chronic administration. In some embodiments, administration can include 1-3 times daily, 1-7 times weekly, or 1-4 times monthly.

[0082] According to some embodiments, administration can include any suitable dosing regimen, depending on, among other things, the medical condition, patient characteristics, route of administration, etc. In some embodiments, dosing via parenteral routes (e.g., injection) can be in an amount ranging from about 1 mg / kg to about 500 mg / kg. In some embodiments, dosing can be in an amount ranging from about 1.5 mg / kg to about 450 mg / kg. In some embodiments, dosing can be in an amount ranging from about 5 mg / kg to about 400 mg / kg. In some embodiments, dosing can be in an amount ranging from about 10 mg / kg to about 350 mg / kg. In some embodiments, dosing can be in an amount ranging from about 20 mg / kg to about 300 mg / kg. In some embodiments, dosing can be in an amount ranging from about 50 mg / kg to about 250 mg / kg. In some embodiments, dosing can be in an amount ranging from about 10 mg / kg to about 200 mg / kg. In some embodiments, dosing via inhalation can be in an amount ranging from about 10 to about 300 mg per dose. In some embodiments, dosage by the inhaled route may be in an amount ranging from about 20 to about 200 mg per dose.

[0083] According to some embodiments, rAAT or compositions comprising it, when used to treat immune-related / inflammatory conditions, may be used in combination with other therapeutic agents. The components of such combinations may be administered sequentially or simultaneously / concurrently in separate or combined pharmaceutical formulations by any suitable administration route.

[0084] According to some embodiments, a method for treating an immune-related condition, such as an inflammatory condition, is provided, comprising administering a therapeutically effective amount of rAAT or a composition comprising same to a subject in need thereof. In some embodiments, rAAT can be administered as a polypeptide or in a suitable pharmaceutical composition.

[0085] According to some embodiments, a therapeutically effective amount refers to an amount sufficient to ameliorate and / or prevent at least one symptom associated with an immune-related disorder or inflammatory condition.

[0086] In the description and claims of this application, the words "include" and "have" and forms thereof are not limited to the members in a list with which the words may be associated. As used herein, the term "comprising" includes the term "consisting only of."

[0087] As used herein, the term "about" can be used to specify a quantity or parameter value (e.g., the length of a member) within a continuous range of values ​​near (and including) a given (stated) value. According to some embodiments, "about" can specify a parameter value to be 80%-120% of the given value. According to some embodiments, "about" can specify a parameter value to be 90%-110% of the given value. According to some embodiments, "about" can specify a parameter value to be 95%-105% of the given value.

[0088] As used herein, according to some embodiments, the terms "substantially" and "about" may be interchangeable.

[0089] While several exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain variations, permutations, additions, and subcombinations thereof. It is therefore intended that the following appended claims and the claims introduced below be interpreted as including all such modifications, permutations, additions, and combinations as fall within their true spirit and scope.

[0090] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Example]

[0091] Example 1: Generation of rAAT-producing cells CHO DG44 cells were transfected under serum-free conditions with a plasmid containing a nucleotide sequence encoding the open reading frame (ORF) of human α1-antitrypsin (represented by SEQ ID NO: 1, which also includes the nucleotide sequence encoding the signal peptide (underlined)). The sequence was codon-optimized for hamster codon usage. The AAT gene was expressed under the strong cytomegalovirus (CMV) promoter. The selection markers were DHFR and neomycin. The plasmid map is shown in Figure 1.

[0092] Cells were optionally further engineered to express hamster β-galactosyl α-2,6 sialyltransferase (ST6Gall) by transfection, which may be under the control of a separate promoter.

[0093] After the initial transfection, each of the eight pools was separated into single cells using FACS and an ultra-high-throughput imaging system was used to verify monoclonality. 4,000 clones were isolated and expanded in 384-, 96-, 24-, 12-, and 6-well plates, followed by expansion in shake flasks. Stepwise selection was performed during expansion. Selection was based on AAT titer determined by ELISA (AAT EISA kit, ICL) and the presence of sialic acid capping by lectin-based assay. The final top 48 clones, proven to be monoclonal, with the best volumetric productivity, specific productivity (PCD), and growth parameters were selected for final evaluation in a bioreactor system (Sartorious GmbH).

[0094] [Table 1]

[0095] [Table 2]

[0096] [Example 2] Purification of rAAT A three-step purification process was used to purify rAAT. Recombinant AAT was purified from a 5-liter cell culture harvest of an AAT-producing cell clone using a multi-step downstream process at laboratory scale. The purification steps were designed to meet the purity, yield, and quality requirements necessary for preclinical evaluation. The purification process is described below and illustrated in Figure 2.

[0097] The culture supernatant was diluted with 20 mM Tris-HCl buffer to a conductivity of 6 mS / cm and filtered through a 0.2 μm filter. The harvest was then captured on a POROS XQ column. After a wash step, bound rAAT was eluted with 20 mM Tris-HCl, 0.4 M NaCl (pH: 7.4 ± 0.1). The pooled elution fractions were diluted with a Tris-HCl-based buffer (20 mM Tris-HCl, pH: 7.2) to a conductivity level of 20 mS / cm and concentrated using a 10 kDa UF membrane. The concentrated material was filtered through a 0.2 μm filter and then loaded onto a multimodal strong anion exchange column, Capto™ Adhere. These columns were run for one cycle in flow-through (non-binding) mode. The material collected in the flow-through was filtered through a 0.2 μm filter. The filtered material was concentrated and diafiltered by UF / DF (10 kDa) against the preformulation solution, 150 mM NaCl. The final preformulation was filtered through a 0.2 μm filter under sterile conditions. Each 5 liter run yielded 7-10 grams of purified AAT, representing a yield of over 70%.

[0098] Typical results of purification from a lab-scale production run are shown in Figures 3A-D. Figure 3A shows the POROS XQ chromatogram, and Figure 3C shows the Capto™ Adhere chromatogram. Correlative pictograms of the SDS-PAGE analysis are shown in Figures 3B and 3D, respectively. rAAT peaks identified in the chromatograms are marked by frames, and the corresponding lanes on the SDS-PAGE gel are marked by arrows. M: Marker; S / St: Standard; H: Recovery; L: Load; U: Unbound; W: Wash; E: Leach (elution); R: Regeneration; F: Flow-through.

[0099] Example 3: Characterization of recombinant AAT The antigenic AAT titer of the starting material (cell culture harvest) was established by turbidimetry and AAT ELISA. Purity was assessed by molecular size distribution (MSD) assay by SEC-HPLC. Figure 4 shows the chromatogram of the final fraction with a monomer peak area of ​​99.2 percent.

[0100] Table 1 below summarizes various properties of rAAT.

[0101] [Table 3]

[0102] Table 2 below provides a summary of the results of characterization of rAAT by LC-MS peptide mapping.

[0103] [Table 4]

[0104] [Example 4 N-glycan profiling by LC-MS analysis] Clone 26 (C-26) and clone 36 (C-36) were selected for further analysis.

[0105] To investigate N-glycan profiling by LC-MS analysis, N-glycans were isolated from various protein samples (plasma-derived AAT or rhAAT clones) and fluorescently labeled with the Waters Glycoworks RapiFluor-MS N-glycan kit (P / N 176003713) using 2-aminobenzamide (2AB) as the fluorescent label according to the manufacturer's instructions.

[0106] A portion of each sample was diluted with PBS to a final concentration of 1 mg / mL. A volume of 15 μL (equivalent to approximately 15 μg) of diluted sample solution was prepared in an Eppendorf tube. Each prepared sample was denatured by adding 7.8 μL of water and 6 μL of 5% RapiGest™ solution (3 mg of RapiGest SF surfactant + 60 μL of 5× Glycoworks Rapid buffer) and incubating at 110°C for 3 minutes. The denatured samples were cooled at room temperature for 3 minutes. N-glycans were released by adding 1.2 μL of GlycoWorks Rapid PNGase F and incubating at 57°C for 5 minutes. The samples were cooled at room temperature for 3 minutes.

[0107] The released N-glycans were labeled with a fluorescent tag by adding 12 μL of RapiFluor-MS labeling solution (9 mg GlycoWorks RapiFluor-MS Reagent Powder + 131 μL GlycoWorks RapiFluor-MS Reagent Solvent) and incubating at room temperature for 5 minutes. After the reaction, the sample was diluted with 358 μL of acetonitrile. The labeled N-glycans were purified by solid-phase extraction using a Waters Glycoworks HILIC μElution plate mounted on a positive pressure device. Wells were conditioned first with 200 μL of HO and then twice with 200 μL of HO / ACN (15 / 85). Each ACN-diluted sample was then loaded and washed twice with 600 μL of HCOOH / HO / ACN (1 / 9 / 90). The N-glycans were finally eluted with three 30 μL portions of Glycoworks SPE elution buffer. Prior to LC-MS analysis, purified samples were diluted with 310 μL of GlycoWorks Sample Diluent (32 / 68 DMF / ACN).

[0108] The labeled N-glycans were analyzed by LC-MS. The LC-MS system used was a Waters UPLC Integrated System Acquity UPLC LCLas with a fluorescence detector online coupled to a Waters Xevo G2-S Q-ToF. Separation was performed using a BEH Glycan Column, 100 mm x 2.1 mm, 1.7 μm (Waters, Milford, MA, Cat. No. 186004742). The solvents used were A: 100% acetonitrile; B: 50 mM ammonium formate, pH 4.4, adjusted with formic acid and filtered through a 0.2 μm pore. The separation gradient was Buffer A, varying from 39% to 47% at a flow rate of 0.5 mL / min over 16 min. The fluorescence detector was set to an excitation wavelength of 360 nm and an emission wavelength of 428 nm using a high-power lamp. The MS was set to positive electrospray MSE scan mode, scanning over the m / z range of 50–4000 over a time range of 10–30 min. UPLC-MS data were processed using validated UNIFI software, version 1.8.2. Relative quantification of each detected glycoform was finally performed within the detected population of N-glycans detected above the 1% reporting threshold.

[0109] The results are shown in Table 3 below, which shows the N-glycan profiles of the C26 and C36 clones of rAAT compared to plasma-derived AAT (pdAAT, Glassia), and more specifically the percentage of fucosylated N-glycans in the various samples.

[0110] [Table 5]

[0111] Thus, the above results clearly demonstrate that the rATT proteins of the present disclosure (i.e., clones 26 and 36) are highly fucosylated (more than 10-fold) compared to plasma-derived AAT protein.

[0112] Example 5: In vitro biological activity of rAAT To test the immunomodulatory effects of the disclosed rAAT, various in vitro studies were performed on mouse splenitis, mouse macrophages, and human peripheral blood mononuclear cells (PBMCs). The studies revealed a surprising enhanced effect of rAAT on the levels of various immune-related markers.

[0113] (I. Mouse splenocytes CXCL1 / KC, TNFα) A study was conducted to evaluate the efficacy of recombinant AAT clones compared with plasma-derived AAT in inhibiting LPS-induced KC (CXCL1) and TNFα secretion in mouse splenocytes. Splenocytes were freshly isolated from C57BL / 6 male mouse spleens, resuspended, and plated in 96-well plates at 5 × 10 5 Cells were seeded at 1000 cells / well. The cells were pre-incubated with different concentrations of pdAAT and different clones of rAAT for 5 hours, supplemented with the same concentration of fresh AAT, and then stimulated with 10 ng / mL LPS (Sigma Cat# L2630) for another 24 hours. The cell supernatant from each well was then collected and evaluated for KC concentration (CXCL1) by specific ELISA (R&D Cat#DY453) and TNFα concentration (R&D Cat#DY410).

[0114] Statistical evaluation of the data was performed using a mixed linear model (LMM), a statistical model that includes both fixed and random effects. Statistical analysis was performed on the four final clones, as well as pdAAT. At all concentrations tested, clone 26 was significantly more potent than the other clones and pdAAT, peaking at 0.5 mg / mL, reducing KC secretion by up to 93% and TNFα secretion by up to 90%.

[0115] As shown in Figure 5A, a significant reduction in LPS-induced KC secretion was identified at all tested concentrations: 0.25–0.5 mg / mL for rAAT clone 36, 0.25–1 mg / mL for rAAT clone 26, and only 4 mg / mL for pdAAT. The maximal effect on KC reduction was observed at 0.25–0.5 mg / mL for rAAT clone 26, with approximately 82–93% reduction, and optimal reduction was achieved at 0.5 mg / mL.

[0116] As shown in Figure 5B , a significant decrease in LPS-induced TNFα secretion was identified at all tested concentrations for both C26 and C36 clones, but the strongest decrease was observed with clone 26 at 0.25–1 mg / mL, showing a reduction of approximately 85–90%.

[0117] (II. TNFα, CXCL1 / KC, and IL-10 in the RAW264.7 murine macrophage cell line) The anti-inflammatory activity of recombinant AAT was also tested in mouse macrophages. Cells were plated in 96-well plates at 5 × 10 5 Cells were seeded at 1000 x g / well. Cells were treated with different concentrations of pdAAT and rAAT and stimulated with 5 ng / ml LPS for 24 hours, or pre-incubated with different concentrations of pdAAT and rAAT for 5 hours, supplemented with the same concentrations of fresh AAT, and then stimulated with 5 ng / ml LPS (Sigma Cat# L2630) for an additional 24 hours. Cell supernatants from each well were then collected and evaluated for KC concentration (CXCL1) by specific ELISA (R&D Cat# DY453), TNFα concentration (R&D Cat# DY410), and IL-10 concentration (R&D Cat# DY417).

[0118] As shown in Figure 6A, a significant reduction in LPS-induced TNFα secretion was identified at both tested concentrations of rAAT. rAAT was significantly more potent than pdAAT, with a TNFα reduction effect of 56–60%.

[0119] As shown in Figure 6B, a significant reduction in LPS-induced CXCL1 secretion was observed at both concentrations of rAAT tested. rAAT was significantly more potent than pdAAT, with a CXCL1 reduction effect of 86–94%.

[0120] Interestingly, as shown in Figure 6C, both concentrations of recombinant AAT increased anti-inflammatory IL-10 secretion by more than 50%, whereas pdAAT had no effect on its secretion.

[0121] (III. Human PBMC IL-8, IL-6, TNFα, IL-10) Human PBMCs were freshly isolated by leukapheresis from healthy donors. Cells were plated in 96-well plates at 5 × 10 5 Cells were seeded at 1000pg / well. The cells were pre-incubated with different concentrations of pdAAT and rAAT for 5 hours, supplemented with the same concentrations of fresh AAT, and then stimulated with 10ng / mL LPS (Sigma Cat# L2630) for another 24 hours. The cell supernatant was then collected from each well, and the concentrations of IL-8, IL-6, IL-10, and TNFα (TNF alpha / TNFα) were determined by specific ELISA.

[0122] The results are shown in Figures 7A-7C. As shown in Figure 7A, both Clone 26 and pdAAT reduced IL-6 secretion by 40-55%. However, while 4 mg / ml of pdAAT was required for 50% inhibition, only 1 / 16 the concentration (0.25 mg / ml) of Clone 26 was required to induce the same effect.

[0123] rAAT clone 26 was also the most potent in inhibiting LPS-induced TNFα secretion in human PBMCs. As shown in Figure 7B, both recombinant clones at 1 mg / ml inhibited TNFα secretion by more than 90%, whereas pdAAT was ineffective even when used at a 4-fold higher concentration.

[0124] Interestingly, dexamethasone (a corticosteroid that blocks the release of inflammatory cytokines) also inhibited anti-inflammatory IL-10 secretion (as shown in Figure 7C), whereas plasma-derived and recombinant AAT increased its secretion in a dose-dependent manner. Although the contribution of pdAAT was not significant at a concentration of 4 mg / ml, both recombinant clones increased IL-10 by more than 50%.

[0125] Example 6: In vivo biological activity of rAAT The biological activity of rAAT in vivo was evaluated in a mouse model of acute myocardial infarction (AMI). Echocardiography was used to assess infarct size and cardiac function as detailed below.

[0126] Adult male CD1 mice (8–10 weeks old) underwent experimental myocardial ischemia / reperfusion (IR) by transient left coronary artery occlusion for 30 minutes, followed by 24 hours of reperfusion. Mice were anesthetized with ketamine / xylazine / acepromazine (100 / 15 / 1 mg / kg) and then orally intubated and connected to a rodent ventilator (Minivent, Harvard Apparatus). After 30 minutes, the arterial occlusion was released to initiate reperfusion. After verification of successful reperfusion, rAAT (C26) or a control treatment was administered by intraperitoneal injection. A sham control was performed without coronary artery occlusion.

[0127] After 24 h, mice were anesthetized with isoflurane (1.5–3%) and subjected to echocardiography to measure left ventricular fractional shortening (LVFS) to calculate left ventricular ejection fraction (LVEF). The results are shown in Figure 8A.

[0128] Viable myocardium was stained using 10% triphenyltetrazolium chloride (TTC, Sigma-Aldrich) in PBS, and non-risk areas were marked using phthalo blue (Heucotech), which is not compromised by ischemic injury. Myocardial damage was also measured by pathological assessment of viability. Infarct size was expressed as a percentage of the total left ventricular (LV) myocardium (Figure 8C) and as a percentage of the area at risk (AAR), as shown in Figure 8B.

[0129] To further assess myocardial injury, plasma obtained from mice was used to measure cardiac troponin I (cTnI) using an ELISA assay (Life Diagnostics). ELISA was also used to detect plasma IL-18 as a measure of inflammasome activation.

[0130] The results clearly demonstrate that treatment of mice with rAAT clone 26 resulted in a significant reduction in infarct size. Compared to vehicle-treated mice, infarct size was reduced by approximately 60-70% in mice treated with a 15 mg / kg dose of rAAT clone 26. A 50% reduction was observed in mice treated with a 5 mg / kg dose, demonstrating a dose-response effect. In addition, treatment with a 15 mg / kg dose of rAAT clone 26 resulted in significant preservation of LV systolic function, measured as LVEF by echocardiography 24 hours after I / R injury, and an increase in infusion rate.

[0131] Example 7 - In vivo biological activity of rAAT in an intestinal ischemia-reperfusion (I / R) injury model Cell culture and animal studies have shown that AAT limits tissue injury and promotes cell and tissue survival. AAT can promote tolerance in animal models by downregulating early inflammation and promoting the induction and stabilization of regulatory T cells. AAT has been shown to reduce intestinal permeability in a mouse model of inflammatory bowel disease

[17] . AAT has shown good results in different animal organ models of IRI in organs: kidney, liver, lung, heart, and brain [2, 18-19]. However, there are no data regarding the effect of AAT on intestinal IRI. As exemplified herein, the in vivo biological activity of rAAT was evaluated in an intestinal ischemia-reperfusion (IRI) model in rats. Survival of rats after IRI was evaluated as detailed below.

[0132] Experiments were performed in a validated rat model of intestinal IRI in which animals were exposed to 60 minutes of intestinal ischemia followed by 7 days of reperfusion and monitored for survival. Experiments were performed in male Sprague-Dawley rats (280-350 grams). Surgery was performed under anesthesia using an intraperitoneal injection of ketamine (100 mg / mL) / xylazine (20 mg / mL) as follows: (1) Standard laparotomy with dissection and clamping of the superior mesenteric artery and assessment of ischemia by paleness of the small bowel and absence of mesenteric vascular pulsation. (2) Temporary abdominal closure with clips. (3) In the 7-day group, after 60 min of warm ischemia, the clamps were removed, 1 mL of 0.9% warm NaCl was administered intraperitoneally (to compensate for fluid loss), and the abdomen and skin were sutured with sutures (Prolene 3.0 and 4.0, respectively).

[0133] In the treatment group, AAT was administered intravenously during ischemia and before reperfusion. The control / sham group received an equivalent volume of NaCl 0.9% only. The sham group underwent identical procedures except for SMA occlusion.

[0134] Buprenorphine (Vetergesic® 0.3 mg / mL) was administered subcutaneously on postoperative days 0, 1 and 2 in the survival group.

[0135] At various post-reperfusion time points (180 min, 24 h, and 7 days), animals were re-anesthetized and euthanized by exsanguination for blood and intestinal sample collection. In the sacrifice group, samples from the terminal ileum were collected and immediately mounted in a 37°C Ussing chamber to measure permeability and functionality. Samples of the jejunum, ileum, and rectum were collected for histopathological evaluation (in 4% formaldehyde) and gene expression analysis (flash-frozen in liquid N2).

[0136] Example 8: In vitro tissue protective activity of rAAT The tissue protective activity of rAAT in vitro was evaluated in a wound healing (gap repair by cell migration) model in A549 lung cancer epithelial cells and human umbilical vein endothelial cells (HUVECs).

[0137] (1. A549 lung cancer epithelial cells) Cells were seeded at 60,000 / well onto 24-well plates in 500 μl medium.

[0138] Mitomycin C was used to inhibit proliferation and distinguish between cell proliferation and migration.

[0139] Gaps were created by straight scratching with a 200 μL pipette tip at a 30° angle, simulating wounding of a confluent cell layer.

[0140] Cells were treated with 1 mg / ml pdAAT or two rAAT clones (C26 or C36) in 2% serum medium. Controls were treated with vehicle (saline).

[0141] Photographs of the cells were taken at 0, 16, and 24 hours. Additionally, wound closure was calculated by ImageJ and presented as the percentage of wound closure after 16 and 24 hours.

[0142] The results are shown in Figures 9A-G and clearly demonstrate the enhanced wound healing effect of the tested rAAT compared to control treatment or pdAAT.

[0143] (2. Human umbilical vein endothelial cells) Cells were seeded into Ibidi culture inserts, which have two compartments separated by a distinct gap, with 21,000 cells seeded into each compartment of the insert. Mitomycin C was used to inhibit proliferation and distinguish between cell proliferation and migration. Cells were treated with 1 mg / ml pdAAT or rAAT in 5% serum medium. Controls were treated with vehicle (saline). Photographs of the cells were taken at time 0, 13 and 18 hours and wound closure was calculated by ImageJ and presented as percentage of wound closure after 13 and 18 hours.

[0144] The results are shown in the graph shown in Figure 9H, which clearly demonstrates that rAAT has an enhanced wound healing effect on endothelial cells (as determined by the percentage of wound closure) compared to the control and pdAAT.

[0145] [reference] 1. Viglio, S., et al., Methods of Purification and Application Procedures of Alpha1 Antitrypsin: A Long-Lasting History. Molecules, 2020. 25(17): p. 4014. 2. Daemen, MA, et al., Functional protection by acute phase proteins α1-acid glycoprotein and α1-antitrypsin against ischemia / reperfusion injury by preventing apoptosis and inflammation. Circulation, 2000. 102(12): p. 1420-1426. 3. Eltzschig, HK and T. Eckle, Ischemia and reperfusion--from mechanism to translation. Nat Med, 2011. 17(11): p. 1391-401. 4. Soares, ROS, et al., Ischemia / Reperfusion Injury Revisited: An Overview of the Latest Pharmacological Strategies. Int J Mol Sci, 2019. 20(20). 5. Kalogeris, T., et al., Cell biology of ischemia / reperfusion injury. Int Rev Cell Mol Biol, 2012. 298: p. 229-317. 6. Virani, S.S., et al., Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation, 2020. 141(9): p. e139-e596. 7. Seropian, I.M., et al., Anti-inflammatory strategies for ventricular remodeling following ST-segment elevation acute myocardial infarction. J Am Coll Cardiol, 2014. 63(16): p. 1593-603. 8. Toldo, S., et al., Alpha-1 antitrypsin inhibits caspase-1 and protects from acute myocardial ischemia-reperfusion injury. J Mol Cell Cardiol, 2011. 51(2): p. 244-51. 9. Mauro, A.G., et al., A preclinical translational study of the cardioprotective effects of plasma-derived alpha-1 anti-trypsin in acute myocardial infarction. Journal of Cardiovascular Pharmacology, 2017. 69(5): p. 273-278. 10. Carden, D.L. and D.N. Granger, Pathophysiology of ischaemia-reperfusion injury. The Journal of Pathology, 2000. 190(3): p. 255-266. 11. Mallick, I.H., et al., Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig Dis Sci, 2004. 49(9): p. 1359-77. 12. Demirkan, A., B. Savaand M. Melli, Endotoxin level in ischemia-reperfusion injury in rats: effect of glutamine pretreatment on endotoxin levels and gut morphology. Nutrition, 2010. 26(1): p. 106-11. 13. Beutler, B. and E.T. Rietschel, Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol, 2003. 3(2): p. 169-76. 14. Swamy, M., et al., Epithelial decision makers: in search of the 'epimmunome'. Nat Immunol, 2010. 11(8): p. 656-65. 15. Kartalija, M., et al., Patients with nontuberculous mycobacterial lung disease exhibit unique body and immune phenotypes. American journal of respiratory and critical care medicine, 2013. 187(2): p. 197-205. 16. Schuster, R., et al., Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action. Cell Immunol, 2020. 356: p. 104177. 17. Collins, C.B., et al., Alpha-1-antitrypsin therapy ameliorates acute colitis and chronic murine ileitis. Inflamm Bowel Dis, 2013. 19(9): p. 1964-73. 18. Ikebe, N., et al., Protective effect of S-nitrosylated alpha(1)-protease inhibitor on hepatic ischemia-reperfusion injury. J Pharmacol Exp Ther, 2000. 295(3): p. 904-11. 19. Moldthan, H.L., et al., Alpha 1-antitrypsin therapy mitigated ischemic stroke damage in rats. J Stroke Cerebrovasc Dis, 2014. 23(5): p. e355-63.

Claims

1. Recombinant human αl-antitrypsin (rhAAT) comprising at least about 75% fucosylated N-linked glycans.

2. 2. The rhAAT of claim 1, comprising at least about 80% fucosylated glycans.

3. 2. The rhAAT of claim 1, comprising at least about 90% fucosylated glycans.

4. The rhAAT of any one of claims 1 to 3, wherein about 80 to more than 95% of the fucose units are core fucose.

5. The rhAAT according to any one of claims 1 to 4, wherein about 90% of the fucose units are core fucose.

6. The rhAAT of any one of claims 1 to 5, wherein about 60-80% of the fucosylated N-linked glycans are diantennary.

7. The rhAAT of any one of claims 1 to 6, wherein about 70% of the fucosylated N-linked glycans are diantennary.

8. The rhAAT of any one of claims 1 to 7, wherein about 5% to 25% of the fucosylated N-linked glycans are triantennary.

9. The rhAAT of any one of claims 1 to 8, wherein about 10% of the fucosylated N-linked glycans are triantennary.

10. The rhAAT of any one of claims 1 to 9, wherein about 5% to 20% of the fucosylated N-linked glycans are tetraantennary.

11. The rhAAT of any one of claims 1 to 10, wherein about 10% of the fucosylated N-linked glycans are tetraantennary.

12. The rhAAT of any one of claims 1 to 11, having increased immunomodulatory activity compared to the immunomodulatory activity of purified plasma-derived AAT (pdAAT).

13. 13. The rhAAT of claim 12, having at least about 5% greater immunomodulatory activity compared to the immunomodulatory activity of purified plasma-derived AAT (pdAAT).

14. The rhAAT of any one of claims 12 to 13, wherein the immunomodulatory activity is characterized by a decrease in the activity, expression and / or secretion levels of one or more of IL-1β, TNFα, IL-6, IL8, IL 18, MCP1 / CCL2.

15. 15. The rhAAT of any one of claims 12 to 14, wherein the immunomodulatory activity is characterized by an increase in the activity, expression and / or secretion levels of IL-10 and / or IL-1 receptor antagonist (IL-IRa).

16. The rhAAT of any one of claims 1 to 15, produced or expressed in a Chinese hamster ovary (CHO) cell line.

17. 17. The rhAAT of claim 16, wherein the CHO cell line is a CHO DG44 cell line.

18. The rhAAT of any one of claims 16 to 17, wherein the CHO cell line further expresses -2,6-sialyltransferase.

19. A pharmaceutical composition comprising the rhAAT of any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. 20. The rhAAT of any one of claims 1 to 18 or the pharmaceutical composition of claim 19 for use in treating or preventing an inflammatory condition in a subject in need thereof.

21. A method for preventing or treating an inflammatory condition in a subject in need thereof, comprising administering a therapeutically effective amount of the rhAAT of any one of claims 1 to 18 or the pharmaceutical composition of claim 19.

22. 22. The method of claim 21, wherein the inflammatory condition is a pulmonary disease selected from the group consisting of alpha-1 antitrypsin deficiency (AATD), small airway disease, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, pneumonia, parenchymal and fibrotic lung diseases or disorders, interstitial pulmonary fibrosis, reinflammation, acute respiratory distress syndrome (ARDS), and sarcoidosis.

23. 23. The method of any one of claims 21-22, wherein the inflammatory condition is selected from the group consisting of graft versus host disease (GVHD), ischemia-reperfusion injury, post-transplant ischemia / reperfusion injury, acute myocardial infarction, acute kidney injury, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease, Crohn's disease, ulcerative colitis, psoriasis, type 1 diabetes, dermatitis, pneumonia, sepsis, wound healing, and systemic lupus erythematosus.

24. The method of any one of claims 21 to 23, wherein the inflammatory condition is ischemia-reperfusion injury.

25. The method of any one of claims 21 to 24, wherein the pharmaceutical composition is administered by injection.

26. 26. The method of claim 25, wherein the pharmaceutical composition is administered at a dose of about 200 mg / kg to about 350 mg / kg.

27. The method of any one of claims 21 to 24, wherein the pharmaceutical composition is administered by inhalation.

28. 28. The method of claim 27, wherein the pharmaceutical composition is administered at a dose of about 20 mg / dose to about 200 mg / dose.

29. 29. The method of any one of claims 21-28, wherein the administration occurs once daily, 1-3 times daily, 1-7 times weekly, 1-4 times monthly, or any combination thereof.

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