An effective amount of recombinant serpin-Fc fusion protein for use in a method of treating a subject with AAT deficiency

Abstract

This application relates to an aqueous solution of an engineered AAT-Fc fusion dimer protein in an effective dosage for use in a method of treating or alleviating a condition associated with abnormal serine protease activity in a subject in need of such a method of treating or alleviating a condition associated with abnormal serine protease activity.

Description

Technical Field

[0001] Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 342,264, filed May 16, 2022, and U.S. Provisional Application No. 63 / 492,692, filed Mar. 28, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference in its entirety. The file name of the XML file containing the Sequence Listing XML is "INHI-044_001WO_SeqList_ST26". The XML file was created on May 8, 2023, and has a size of 4.90 kilobytes.

[0003] Technical Field The present invention relates to an aqueous solution comprising an effective amount range of a human AAT-IgG Fc fusion protein for treating AAT deficiency and an administration interval of the AAT-IgG Fc fusion protein.

Background Art

[0004] Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed genetic disorder that affects an estimated 100,000 patients in the United States. It is characterized by insufficient levels of AAT that cause emphysema, loss of lung function, and reduced life expectancy. Based on biochemical efficacy, plasma-derived AAT (pdAAT) therapy was approved in the 1980s and is administered weekly to maintain serum AAT concentrations above 11 μM, which is below the normal range. Additionally, AAT therapy has been reported to downregulate inflammation and is being studied to suppress inflammatory diseases and disorders. Since then, there has been little progress with new therapies, and the cost / supply of pdAAT remains an issue. A wide range of engineered antibody proteins, including bispecific and trispecific antibodies, have been developed. Some engineered proteins have also been developed in which the Fc, separated from the Fab portion of the antibody molecule (the portion that confers antigen-binding specificity), serves a purpose other than its physiological one, particularly to extend the in vivo half-life of the engineered protein. WO 2013 / 003641 A2 and WO 2016 / 069574 A1 disclose engineered human immunoglobulin G (human IgG) fusion proteins that include a serpins polypeptide or an amino acid sequence derived from a serpin. It is necessary to determine the dosing regimen of these engineered proteins for effective treatment and half-life extension in a subject system. The present invention addresses this need. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] The present disclosure provides a method of treating or alleviating symptoms associated with abnormal serine protease activity in a subject in need thereof, the method comprising administering to the subject an AAT-Fc fusion protein by infusion at a dose of about 10 to 120 mg / Kg on the first day of treatment and then every 3 or 4 weeks, wherein the AAT-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or comprises the AAT polypeptide of SEQ ID NO: 2 and the Fc polypeptide of SEQ ID NO: 3.

[0006] In some embodiments, the method includes administering a dose of about 40 to 120 mg / Kg. In some embodiments, the method includes administering a dose of about 40 to 80 mg / Kg.

[0007] In some embodiments, the method includes administering a dose of about 60 to 120 mg / Kg.

[0008] In some embodiments, the method includes administering a dose of about 80 mg / Kg.

[0009] In some embodiments, the method includes administering a dose of about 120 mg / Kg.

[0010] In some embodiments, subsequent doses are higher than previous doses. In some embodiments, subsequent doses are lower than previous doses. In some embodiments, subsequent doses are the same as previous doses.

[0011] In some embodiments, the method includes administering a dose of about 120 mg / Kg on the first day of treatment and every three weeks thereafter.

[0012] In some embodiments, the method includes administering a dose of about 120 mg / Kg on the first day of treatment and every four weeks thereafter.

[0013] In some embodiments, the method comprises: (a) determining the level of serine protease expression or activity in a subject prior to administration of a first dose to obtain a baseline of expression or activity; (b) determining the level of serine protease expression or activity in the subject for a period of at least 3 weeks after administration of the first dose or a subsequent dose; and (c) if the serine protease expression or activity in the subject is above the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) that is greater than a previous dose; or (d) if the serine protease expression or activity in the subject is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is lower than a previous dose.

[0014] In some embodiments, the method comprises: (a) determining the level of AAT expression or activity in a subject prior to administration of a dose to obtain a baseline of expression or activity; (b) determining the level of AAT expression or activity in the subject for a period of at least 3 weeks after administration of the dose; and (c) if the AAT expression or activity in the subject is above the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) that is greater than a previous dose; or (d) if the AAT expression or activity in the subject is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is lower than a previous dose.

[0015] In some embodiments, the method comprises: (a) determining the serum AAT level in a subject for a period of at least 3 weeks after administration of a first dose or a subsequent dose of an AAT-Fc fusion protein to obtain the serum AAT level; (b) administering a subsequent dose of the AAT-Fc fusion protein that is greater than or equal to the previous dose of the AAT-Fc fusion protein if the serum AAT level in the subject is below the normal range; or (c) administering a subsequent dose of the AAT-Fc fusion protein that is lower than the previous dose if the serum AAT level in the subject is higher than the normal range. In some embodiments of the above method, the functional AAT level is determined. In some embodiments of the above method, the serum AAT level in the subject is less than 15 μM or greater than 50 μM.

[0016] In some embodiments, the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 5 mM Tris; about 150 mM trehalose; about 100 mM sucrose; about 100 mM proline; about 2 mM methionine; and about 0.1% (w / v) poloxamer; wherein the pH of the aqueous solution is adjusted to about 7.3 using either hydrochloric acid or sodium hydroxide; and the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is about 4.3 mM.

[0017] In some embodiments, the AAT-Fc fusion protein is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20. In some embodiments, the pH of the aqueous solution is about 7.0.

[0018] In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0019] In some embodiments, the subject in need of the method has abnormal serine protease activity associated with a disease or disorder selected from the following: AAT deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing diseases or disorders, systemic lupus erythematosus, and multiple sclerosis.

[0020] In some embodiments, the subject has an infection selected from a bacterial infection, a fungal infection, or a viral infection.

[0021] In some embodiments, the subject is human.

[0022] In some embodiments, the infusion is delivered over a period of about 30 to 120 minutes. In some embodiments, the infusion is delivered over a period of about 30 to 60 minutes.

[0023] The present disclosure also provides a unit dose vial comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20, optionally having a pH of 7.0.

[0024] In some embodiments, the unit dose vial comprises an AAT-Fc fusion protein at about 50 mg / ml.

[0025] In some embodiments of the aqueous solution of the present disclosure for use according to any of the methods of the present disclosure, the subject is human.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In practicing the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and compositions are described below. All publications, patent applications, patents, and other references mentioned herein are hereby expressly incorporated by reference in their entirety, but in particular, U.S. Provisional Patent Application No. 63 / 342,264, filed on May 16, 2022; and U.S. Provisional Patent Application No. 63 / 492,692, filed on March 28, 2023 are hereby incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

[0027] Other features and advantages of the present invention will become apparent from and be encompassed by the following detailed description and claims.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

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Figure 4

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure is based in part on the surprising discovery that administration of an α1-antitrypsin (AAT) Fc fusion protein, also referred to herein as INBRX-101, to an individual having α1-antitrypsin deficiency (AATD) results in the restoration of α1-antitrypsin to normal levels.

[0030] AATD is a genetic disorder characterized by insufficient levels of AAT that cause emphysema, loss of lung function, and reduced life expectancy. Current augmentation therapy with plasma-derived AAT requires weekly IV administration due to its short half-life and aims to maintain patients above a serum AAT target concentration of 11 μM. In contrast, INBRX-101 is an engineered recombinant human AAT-Fc fusion protein that has been demonstrated to achieve serum levels of AAT in the normal range exceeding 20 μM over an administration interval of at least 3 weeks. INBRX-101 is the first ATT-Fc fusion protein to use an extended administration interval to achieve and maintain serum AAT levels within the normal range (above 20 μM).

[0031] The extended administration interval of INBRX-101 reduces the frequency of infusions, eliminates the decline in lung function from AATD, and significantly improves the quality of life of patients. Accordingly, the present disclosure provides a method of treating or alleviating symptoms associated with abnormal serine protease activity in a subject by administering to the subject an AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) at a dose of about 10 - 120 mg / Kg by infusion on the first day of treatment and then every 3 or 4 weeks thereafter. In some embodiments, serum AAT levels (e.g., functional AAT levels) are generally maintained above 20 μM between administrations.

[0032] The amino acid sequence of the INBRX-101 monomer is shown below. The Met351Glu mutation in the AAT polypeptide portion of INBRX-101 (SEQ ID NO: 2) is underlined in bold and italics, and the Met358Leu mutation is in bold and italics. The IgG4-Fc polypeptide portion of INBRX-101 (SEQ ID NO: 3) is in italics, and the mutations S228P, L235E, M252Y, and M428L are shown in squares. The GS linker connecting the AAT polypeptide portion and the IgG4-Fc polypeptide portion is shown in bold.

Chemical formula

[0033] INBRX-101 is produced as a monomer but may form a dimer in an aqueous solution. In some embodiments, the INBRX-101 disclosed herein can exist as a mixture of monomeric and dimeric proteins. In some embodiments, the monomers of the dimeric protein may be linked to each other by disulfide bridges. In particular, a pair of immunoglobulin Fc polypeptides or polypeptides derived from immunoglobulin Fc polypeptides are linked as such to form a functional Fc domain.

[0034] In some embodiments, the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is administered at a dose (first or subsequent) of about 40 - 120 mg / Kg (e.g., about 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 105, 105 - 110, 110 - 115 or 115 - 120 mg / Kg). In some embodiments, the dose is about 40 - 80 mg / Kg (e.g., about 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75 or 75 - 80 mg / Kg). In some embodiments, the dose is about 60 - 120 mg / Kg (e.g., about 60 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 105, 105 - 110, 110 - 115 or 115 - 120 mg / Kg). In some embodiments, the dose is about 60 - 80 mg / Kg (about 60 - 65, 65 - 70, 70 - 75 or 75 - 80 mg / Kg).

[0035] In some embodiments, the dose is about 40 mg / Kg. In some embodiments, the dose is about 80 mg / Kg. In some embodiments, the dose is about 120 mg / Kg.

[0036] In some embodiments, a dose of about 80 mg / Kg is administered on the first day of treatment and every three weeks thereafter.

[0037] In some embodiments, a dose of about 80 mg / Kg is administered on the first day of treatment and then every four weeks thereafter.

[0038] In some embodiments, a dose of about 120 mg / Kg is administered on the first day of treatment and then every three weeks thereafter.

[0039] In some embodiments, a dose of about 120 mg / Kg is administered on the first day of treatment and then every four weeks thereafter.

[0040] In some embodiments, subsequent doses are higher than the previous dose. In some embodiments, subsequent doses are lower than the previous dose. In other embodiments, subsequent doses are the same as the previous dose.

[0041] In some embodiments, the period between each administration is the same.

[0042] In some embodiments, each subsequent dose is administered three weeks after the previous dose.

[0043] In some embodiments, each subsequent dose is administered four weeks after the previous dose.

[0044] In some embodiments, one or more subsequent doses are administered three weeks after the previous dose, and then one or more additional doses are administered four weeks after the previous dose.

[0045] The AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is administered by injection, i.e., intravenously.

[0046] In certain embodiments, the AAT-Fc fusion protein is diluted in an infusion bag containing a suitable diluent (e.g., physiological saline, aqueous dextrose solution, etc.). Since an infusion reaction or allergic reaction may occur, a pre-medication to prevent such an infusion reaction is recommended, and prophylactic measures for anaphylaxis should be observed during antibody administration. In certain embodiments, the infusion should be administered to the subject over a period of about 30 minutes to about 4 hours. In certain embodiments, an IV infusion is delivered over a period of about 30 - 240 minutes, about 30 - 180 minutes, about 30 - 120 minutes, or about 30 - 90 minutes, or over a period of about 30 - 60 minutes, or over a shorter period, if the subject does not exhibit signs or symptoms of a harmful infusion reaction. In one embodiment, the IV infusion is delivered over a period of about 30 - 60 minutes. In another embodiment, the infusion is delivered over a period of about 35 - 55 minutes. In another embodiment, the IV infusion is delivered over a period of about 45 minutes.

[0047] Generally, in the above embodiments, the administration is performed at a predetermined frequency or periodicity, or within about 1 - 3 days of such a scheduled interval, and the administration is, for example, once every 3 weeks (±3 days), 1 - 3 days before, 1 - 3 days after, or on the day of the scheduled administration.

[0048] In some embodiments, the method further comprises: (a) determining the level of serine protease expression or activity in a subject prior to administration of a first dose to obtain a baseline of expression or activity; (b) determining the level of serine protease expression or activity in the subject for at least 3 weeks after administration of said dose in the method of the present disclosure; and (c) administering a subsequent dose of the AAT-Fc fusion protein that is greater than the previous dose of the serine protease fusion protein if the serine protease expression or activity in the subject is above the baseline level obtained in step (a); or (d) administering a subsequent dose of the AAT-Fc fusion protein that is lower than the previous dose if the serine protease expression or activity in the subject is lower than the baseline level obtained in step (a).

[0049] In some embodiments, the serine protease activity of the subject in step (a) of the methods disclosed herein is higher than the physiological level of serine protease activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0050] In some embodiments, the serine protease activity of the subject in step (a) of the methods disclosed herein is at least about 1.5 to 2 times higher than the physiological level of serine protease activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0051] Symptoms of abnormal serine protease expression or activity, such as a decrease from normal AAT levels, can include, for example, shortness of breath, excessive coughing with phlegm / sputum production, wheezing, increased chest pain during breathing, decreased exercise capacity, and a persistent low energy state or fatigue.

[0052] In some embodiments, the serine protease activity of the subject can be determined by any conventional method for detecting protein / enzyme activity in the subject's tissue, such as kinetic fluorescence assays, spectrophotometric enzyme assays, calorimetric enzyme assays, light scattering enzyme assays, and microscale thermophoresis.

[0053] In some embodiments, the serine protease expression of the subject in step (a) of the methods disclosed herein is higher than the physiological level of serine protease activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0054] In some embodiments, the serine protease expression of the subject in step (a) of the methods disclosed herein is at least about 1.5 to 2 times higher than the physiological level of serine protease activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity. The normal physiological level of neutrophil elastase (NE), an exemplary serine protease in human plasma, is about 32 - 56 μg / L. In some embodiments, in a subject having abnormal serine protease expression, the level of NE, an exemplary serine protease, can be at least about 48 - 112 μg / L (e.g., about 48 - 52, 52 - 56, 56 - 60, 60 - 66, 66 - 70, 70 - 74, 74 - 78, 78 - 82, 82 - 86, 86 - 90, 90 - 96, 96 - 100, 100 - 104, 104 - 108 or 108 - 112 μg / L).

[0055] In some embodiments, the serine protease expression of the subject can be determined by any conventional method for detecting protein / enzyme expression in the subject's tissue, such as electrochemiluminescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, mass spectrometry, etc.

[0056] Serine proteases are catalytic enzymes produced by the liver in response to pathological conditions such as infectious diseases. α1-Antitrypsin (AAT) is a serine protease inhibitor that regulates the activity of serine proteases, such as neutrophil elastase (NE). Thus, the level of serine protease activity is inversely proportional to the level of AAT expression and activity. A subject having serine protease activity higher than the physiological level of serine protease activity in a normal subject without any symptoms or disorders associated with abnormal serine protease expression or activity may also have AAT lower than the physiological level of serine protease activity in a normal subject without any symptoms or disorders associated with abnormal serine protease expression or activity.

[0057] In some embodiments, the method comprises: (a) determining the level of AAT expression or activity in a subject prior to administration of a first dose to obtain a baseline of expression or activity; (b) determining the level of AAT expression or activity in the subject for a period of at least 3 weeks after administration of said dose in the method of the present disclosure to obtain a baseline level of AAT expression or activity; and (c) administering a subsequent dose of an AAT-Fc fusion protein that is greater than or equal to a previous dose of the AAT-Fc fusion protein if the AAT expression or activity in the subject is below the baseline level obtained in step (a); or (d) administering a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is lower than a previous dose if the AAT expression or activity in the subject is higher than the baseline level obtained in step (a).

[0058] In some embodiments, the method comprises: (a) determining the serum AAT level in a subject for a period of at least 3 weeks after administration of a first dose or a subsequent dose of an AAT-Fc fusion protein to obtain the serum AAT level; (b) administering a subsequent dose of an AAT-Fc fusion protein that is greater than or equal to a previous dose of the AAT-Fc fusion protein if the serum AAT level in the subject is below the normal range; or (c) administering a subsequent dose of an AAT-Fc fusion protein that is lower than a previous dose if the serum AAT level in the subject is higher than the normal range. In some embodiments of the above method, a functional AAT level is determined. In some embodiments of the above method, the serum AAT level in the subject is less than 15 μM or greater than 50 μM.

[0059] In some embodiments, the AAT activity of the subject in step (a) of the method disclosed herein is lower than the physiological level of AAT activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0060] In some embodiments, the AAT activity of the subject in step (a) of the method disclosed herein is at least about 1.5 to 2 times lower than the physiological level of AAT activity in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0061] In some embodiments, the AAT expression of the subject in step (a) of the method disclosed herein is lower than the physiological level of AAT expression in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity.

[0062] In some embodiments, the AAT expression of the subject in step (a) of the method disclosed herein is at least about 1.5 to 2 times lower than the physiological level of AAT expression in a normal subject without symptoms or disorders associated with abnormal serine protease expression or activity. The normal physiological level of AAT in human plasma is about 20 - 48 μM (80 - 220 mg / dL). In some embodiments, the level of AAT in the plasma of the subject is about 10 - 32 μM (e.g., about 10 - 12, 12 - 14, 14 - 16, 16 - 18, 18 - 20, 20 - 22, 22 - 24, 24 - 26, 26 - 28, 28 - 30, 30 - 32 μM). Alpha-1 antitrypsin deficiency (AATD) is associated with plasma concentrations of less than 20 μM (e.g., less than 19 - 20, 18 - 19, 17 - 18, 16 - 17, 15 - 16, 14 - 15, 13 - 14, 12 - 13, 11 - 12, or less than 11 μM). In some embodiments, the subject has severe AAT deficiency if it has been demonstrated to have plasma levels of 11 μM or less.

[0063] In some embodiments, the AAT activity of the subject can be determined by any conventional method for detecting protein / enzyme activity in the tissue of the subject, such as kinetic fluorescence assays, spectrophotometric enzyme assays, calorimetric enzyme assays, light scattering enzyme assays, human neutrophil elastase capacity (ANEC) assays, and microscale thermophoresis, etc. (see, for example, Engelmaier A, Weber A. (2022) J Pharm Biomed Anal.;209:114476).

[0064] In some embodiments, the AAT expression of the subject can be determined by any conventional method for detecting protein / enzyme expression in the subject's tissue, such as electrochemiluminescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, mass spectrometry, and the like.

[0065] In some embodiments, the expression / activity of either or both of AAT or serine protease can be determined in the subject's tissues and body fluids, including blood, serum, plasma, sputum, urine, feces, bronchoalveolar lavage fluid, vaginal lavage fluid, semen, and the like.

[0066] In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 40 - 80 mg / Kg (e.g., 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, or 75 - 80 mg / Kg), and the subsequent dose of the AAT-Fc fusion protein is about 80 - 120 mg / kg (e.g., 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 105, 105 - 110, 110 - 115, or 115 - 120 mg / Kg). In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 40 mg / Kg, and the subsequent dose of the AAT-Fc fusion protein is about 80 mg / Kg. In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 80 mg / Kg, and the subsequent dose of the AAT-Fc fusion protein is about 120 mg / Kg.

[0067] In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 40 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 40 mg / kg. In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 80 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 80 mg / kg. In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is greater than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 120 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 120 mg / kg.

[0068] In some embodiments of a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) that is less than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 80 to about 120 mg / Kg (e.g., 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 105, 105 - 110, 110 - 115, or 115 - 120 mg / Kg) and the subsequent dose of the AAT-Fc fusion protein is about 40 to about 80 mg / kg (e.g., 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, or 75 - 80 mg / Kg).

[0069] In some embodiments of a subsequent dose of an AAT-Fc fusion protein that is lower than a previous dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1), the first or initial dose of the AAT-Fc fusion protein is about 80 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 40 mg / kg. In some embodiments of a subsequent dose of an AAT-Fc fusion protein that is lower than a previous dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1), the first or initial dose of the AAT-Fc fusion protein is about 120 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 80 mg / Kg. In some embodiments of a subsequent dose of an AAT-Fc fusion protein that is lower than a previous dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1), the first or initial dose of the AAT-Fc fusion protein is about 120 mg / Kg and the subsequent dose of the AAT-Fc fusion protein is about 40 mg / Kg.

[0070] In some embodiments, the subsequent dose is administered every 3 weeks, every 4 weeks, every 5 weeks, every 7 weeks, every 8 weeks (or 2 months), every 10 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks (or 4 months), every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks (or 6 months), every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, every 26 weeks, every 27 weeks, every 28 weeks, every 29 weeks, every 30 weeks, every 31 weeks, every 32 weeks (or 8 months), every 33 weeks, every 34 weeks, every 35 weeks, every 36 weeks, every 37 weeks, every 38 weeks, every 39 weeks, every 40 weeks, every 41 weeks, every 42 weeks, every 43 weeks, every 44 weeks, every 45 weeks, every 46 weeks, after the first dose or previous dose, every 47 weeks or every 48 weeks (or 12 months), after the first dose or previous dose.

[0071] In some embodiments, the subsequent dose is administered every 3 weeks after the first or previous dose. In some embodiments, the subsequent dose is administered every 4 weeks after the first or previous dose.

[0072] In some embodiments, the subject in need of the method has abnormal serine protease activity associated with a disease or disorder selected from the following: AAT deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing diseases or disorders, systemic lupus erythematosus, and multiple sclerosis.

[0073] In some embodiments, the subject has an infection selected from a bacterial infection, a fungal infection, or a viral infection. In some embodiments, the subject is a mammal. For use according to any of the methods of the present disclosure or in some embodiments of use, the subject is a human, rodent, cat, dog, cow, horse, camel, or mammalian subject. In some embodiments of use according to any of the methods of the present disclosure, the subject is a human.

[0074] Pharmaceutical composition The engineered AAT-Fc fusion protein of the present invention (e.g., INBRX-101; SEQ ID NO: 1) can be further incorporated into a pharmaceutical composition suitable for administration. Such compositions typically include an AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences, a standard reference text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. The use thereof in the compositions is contemplated, except in cases where any conventional media or agent is incompatible with the active compound. Auxiliary active compounds can also be incorporated into the compositions.

[0075] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, for example, intravenous, epidural, subcutaneous, intramuscular, intradermal, subcutaneous and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, physiological saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate, and agents for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules made of glass or plastic, disposable syringes or multi-dose vials.

[0076] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR® EL (CrEL) (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it is easily injectable. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is considered preferable to include in the composition isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol, sodium chloride. Inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition can result in prolonged absorption of the injectable composition.

[0077] A sterile injectable solution can be prepared by incorporating an AAT fusion protein (e.g., INBRX-101; SEQ ID NO:1) in a suitable solvent in the required amounts, optionally together with one or a combination of components herein, followed by filtration sterilization. Generally, a dispersion is prepared by incorporating an AAT fusion protein (e.g., INBRX-101; SEQ ID NO:1) into a sterile vehicle containing a basic dispersion medium and the required other components from those listed herein. In the case of a sterile powder for preparing a sterile injectable liquid preparation, the preparation method is vacuum drying and lyophilization, which result in a powder of the active ingredient and any desired additional ingredients from its pre-sterilized filtered solution.

[0078] In some embodiments, the pharmaceutical composition comprises unit dose vials. In some embodiments, the unit dose vial comprises an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO:1 at about 5 mg / ml to about 100 mg / ml; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20.

[0079] In some embodiments, the unit dose vial contains an AAT-Fc fusion protein (i.e., INBRX-101; SEQ ID NO: 1) at about 5 mg / ml to about 100 mg / ml, about 9.5 mg / ml of disodium phosphate heptahydrate; about 0.2 mg / ml of monosodium phosphate monohydrate; about 7.26 mg / ml of sodium chloride; about 20.0 mg / ml of trehalose dihydrate; and about 0.1 mg / ml of polysorbate 20 solution. In some embodiments, the unit dose vial contains an AAT-Fc fusion protein at about 5 mg / ml to about 100 mg / ml that comprises the amino acid sequence of SEQ ID NO: 1; about 9.5 mg / ml of disodium phosphate heptahydrate; about 2.0 mg / ml of monosodium phosphate monohydrate; about 7.26 mg / ml of sodium chloride; about 20.0 mg / ml of trehalose dihydrate; and about 0.1 mg / ml of polysorbate 20 solution. In some embodiments, the unit dose vial contains an AAT-Fc fusion protein at about 50 mg / ml. In some embodiments, the contents of the unit dose vial have a pH of about 7.0.

[0080] The pharmaceutical composition comprises an AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) in an aqueous solution having a pH in the range of 7.0 to 8.0 and one or more buffers having at least one ionizable group at a concentration of about 1 to 50 mM; non-charged tonicity modifiers each at 50 to 200 mM; a surfactant at about 0.01 to 2 mg / ml; and optionally, one or more neutral amino acids each at 0 to 300 mM.

[0081] The aqueous solution has a pKa in the range of 4.0 to 10.0, and the pKa is within 2 pH units of the pH of the aqueous solution.

[0082] The total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is less than 30 mM.

[0083] Exemplary buffers include citrate, histidine, maleate, sulfite, aspartame, aspartate, glutamate, tartrate, adenine, succinate, ascorbate, benzoate, phenylacetate, gallate, cytosine, p-aminobenzoic acid, sorbate, acetate, propionate, alginate, urate, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-imino diacetic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, piperazine, N,N'-bis(2-ethanesulfonic acid), phosphate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)glycine and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and their salts, disodium phosphate heptahydrate and monosodium phosphate monohydrate or combinations thereof.

[0084] Exemplary non-charged tonicity adjusters include polyols, sugars (e.g., monosaccharides and disaccharides) and sugar alcohols. In some embodiments, the non-charged tonicity adjuster is selected from the group consisting of glycerol, 1,2-propanediol, mannitol, sorbitol, glucose, sucrose, trehalose, PEG300 and PEG400.

[0085] The total concentration of the non-charged tonicity adjuster, or a combination of two or more tonicity adjusters, is 50-1000 mM, such as 200-600 mM, 200-500 mM, or the total concentration of the non-charged tonicity adjuster, or a combination of two or more tonicity adjusters, is 50-500 mM, such as 100-400 mM, 150-350 mM, 200-300 mM, or about 250 mM. In some embodiments, the total concentration of the non-charged tonicity adjuster or a combination of two or more tonicity adjusters is 50-150 mM.

[0086] In some embodiments, the aqueous solution contains one or more neutral amino acids selected from glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine.

[0087] In some embodiments, the total concentration of one or more neutral amino acids in the aqueous solution is 2 to 100 mM. In some embodiments, the total concentration of one or more neutral amino acids in the aqueous solution is 20 to 600 mM, such as 20 to 500 mM, such as 20 to 400 mM, such as 20 to 300 mM, such as 50 to 300 mM. In some embodiments, the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is less than 20 mM.

[0088] As used herein, a neutral amino acid is an amino acid whose side chain does not contain ionizable groups that are significantly ionized at the pH of the aqueous solution (e.g., more than 20%, especially more than 50% of the side chains have a negative or positive charge). Exemplary neutral amino acids are glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine, especially their L-isomers.

[0089] In some embodiments, the aqueous solution contains a nonionic surfactant. In some embodiments, the nonionic surfactant is selected from the group consisting of alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol (poloxamers), and alkyl phenyl ethers of polyethylene glycol. In some embodiments, the nonionic surfactant is a polysorbate such as polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a block copolymer of polyethylene glycol and polypropylene glycol (poloxamer), such as poloxamer 188.

[0090] In some embodiments, the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is less than 20 mM.

[0091] In some embodiments, the pH of the aqueous solution is from about 7.2 to about 7.5 (e.g., 7.2, 7.3, 7.4, or 7.5).

[0092] In some embodiments, the aqueous solution contains a nonionic surfactant. In some embodiments, exemplary nonionic surfactants include alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol (poloxamers), and alkyl phenyl ethers of polyethylene glycol. In some embodiments, the exemplary nonionic surfactant includes a polysorbate such as polysorbate 20 or polysorbate 80. In some embodiments, the exemplary nonionic surfactant includes a block copolymer of polyethylene glycol and polypropylene glycol (poloxamer), such as poloxamer 188. In some embodiments, the nonionic surfactant is present at a concentration of from about 0.1 mg / ml to about 10 mg / ml (e.g., about 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, or 10 mg / ml).

[0093] Polysorbate 20 is a monoester formed from lauric acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 80 is a monoester formed from oleic acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 20 is known by various trade names, including especially Tween 20 and also Alkest TW20. Polysorbate 80 is known by various trade names, including especially Tween 80 and also Alkest TW80. Other suitable polysorbates include polysorbate 40 and polysorbate 60.

[0094] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 5 mM tris; about 150 mM trehalose; about 100 mM sucrose; about 100 mM proline; about 2 mM methionine; and about 1 mg / ml (0.1% (w / v)) poloxamer; where the pH of the aqueous solution is adjusted to about 7.3 using either hydrochloric acid or sodium hydroxide; and the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is about 4.3 mM. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0095] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 0.6 mg / ml of Tris; about 51.3 mg / ml of trehalose; about 34.23 mg / ml of sucrose; about 11.5 mg / ml of proline; about 0.3 mg / ml of methionine; and about 1 mg / ml (0.1% (w / v)) of poloxamer; wherein the pH of the aqueous solution is adjusted to about 7.3 using either hydrochloric acid or sodium hydroxide; and the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is about 4.3 mM. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0096] In some embodiments, the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 0.54 mg / ml of Tris base; about 0.716 mg / ml of Tris hydrochloride 21; about 56.7 mg / ml of trehalose dihydrate; about 34.2 mg / ml of sucrose; about 11.5 mg / ml of proline; about 0.3 mg / ml of methionine; and about 1 mg / ml (0.1% (w / v)) of poloxamer; wherein the pH of the aqueous solution is adjusted to about 7.3 using either hydrochloric acid or sodium hydroxide; and the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is about 4.3 mM. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0097] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; one or more buffers each being a substance at 1 to 40 mM; one or more tonicity modifiers each at 50 to 100 mM; a surfactant at about 1 mg / ml; and a solvent. In some embodiments, the aqueous solution comprises an AAT-Fc fusion protein at about 50 mg / ml.

[0098] In some embodiments, the AAT-Fc fusion protein is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; sodium phosphate at about 50 mM; sodium chloride at about 125 mM; trehalose dihydrate at about 2% (w / v); and polysorbate 20 at about 0.1 mg / ml. In some embodiments, the pH of the aqueous solution is about 7.0. In some embodiments, the aqueous solution comprises an AAT-Fc fusion protein at about 50 mg / ml.

[0099] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: disodium phosphate heptahydrate at about 35.4 mM, monosodium phosphate monohydrate at about 1.7 mM, sodium chloride at about 124.2 mM, trehalose dihydrate at about 52.9 mM, and polysorbate 20 at about 0.1 mg / ml. In some embodiments, the aqueous solution comprises an AAT-Fc fusion protein at about 50 mg / ml.

[0100] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: disodium phosphate heptahydrate at about 35.4 mM, monosodium phosphate monohydrate at about 15.6 mM, sodium chloride at about 124.3 mM, trehalose dihydrate at about 52.9 mM, and polysorbate 20 at about 0.01% (w / v). In some embodiments, the aqueous solution comprises an AAT-Fc fusion protein at about 50 mg / ml.

[0101] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 9.5 mg / ml of disodium phosphate heptahydrate; about 0.2 mg / ml of monosodium phosphate monohydrate; about 7.26 mg / ml of sodium chloride; about 20.0 mg / ml of trehalose dihydrate; and about 0.1 mg / ml of polysorbate 20. In some embodiments, the pH of the aqueous solution is 7.0. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0102] In some embodiments, the AAT-Fc fusion protein is in an aqueous solution comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 9.5 mg / ml of disodium phosphate heptahydrate; about 2.0 mg / ml of monosodium phosphate monohydrate; about 7.26 mg / ml of sodium chloride; about 20.0 mg / ml of trehalose dihydrate; and about 0.1 mg / ml of polysorbate 20 solution. In some embodiments, the pH of the aqueous solution is about 7.0. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0103] Note that all references to "pH" herein refer to the pH of an aqueous solution evaluated at 25°C. All references to "pKa" refer to the pKa of an ionizable group evaluated at 25°C (see CRC Handbook of Chemistry and Physics, 79th Edition, 1998, D.R. Lide). If necessary, the pKa values of the amino acid side chains present in the polypeptide can be estimated using an appropriate computer.

[0104] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution having an osmolality that is physiologically acceptable and thus suitable for parenteral administration. Accordingly, the osmolality of the aqueous solution is suitably 200 - 500 mOsm / L, for example about 300 mOsm / L. The aqueous solution is, for example, isotonic with human plasma. In other embodiments, the osmolality of the aqueous solution is 300 - 500 mOsm / L, for example, about 400 - 460 mOsm / L. The aqueous solution may also be hypotonic or hypertonic, for example, intended for dilution prior to administration.

[0105] In some embodiments, the aqueous solution may further contain a preservative such as a phenolic or benzyl preservative. Exemplary preservatives are suitably selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propyl paraben and methyl paraben, particularly phenol, m-cresol and benzyl alcohol. The concentration of the preservative is typically 10 - 100 mM, for example 20 - 80 mM, for example 25 - 50 mM. The optimal concentration of the preservative in the aqueous solution is selected to ensure that the aqueous solution passes the Pharmacopeial Antimicrobial Effectiveness Test (USP <51>, Vol. 32).

[0106] In one embodiment, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation including an implant and a microencapsulation delivery system. Exemplary biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0107] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the composition in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for the treatment of individuals.

[0108] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.

[0109] The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention described in the claims.

[0110]

Table 1

[0111] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Examples

[0112] General method Detection and quantification of recombinant human AAT-Fc fusion protein INBRX-101 The detection and quantification of INBRX-101 in human serum were performed using an electrochemiluminescence (ECL) assay in which anti-α-1 antitrypsin (ANTI-AAT CX2115, InhibRx, #2019011) was coated onto an MSD high-binding plate (ECL-compatible). INBRX-101 in standards, QCs, controls, and samples was captured onto the coated plate. After thoroughly washing the wells to remove unbound compound, ruthenium-conjugated anti-IgG4 (Syneos Health, #94589) was added to the wells. The conjugate binds to the captured INBRX-101. After incubation with the detection reagent, the plate was washed and subsequently MSD read buffer was added. The assay plate was then read using an MSD ECL plate reader. The electrochemiluminescence signal generated was proportional to the amount of INBRX-101 present in the standards, quality control samples (QCs), controls, and samples tested. The concentration of INBRX-101 was back-calculated from the non-linear regression of the standards. Data were acquired using a Meso Scale Discovery (MSD) Sector S 600. Data capture and analysis were performed using SoftMax Pro Software, version 5.2 or 7.0.1, SoftMax Pro Protocol TM2381.00.

[0113] All calculated values, including but not limited to mean concentration, SD, %CV, and %RE, are reported to three significant digits. Overall statistical calculations (SD, %CV, and %RE) are performed using concentration values rounded to three significant digits.

[0114] Assay for the quantification of the function of INBRX-101 and endogenous AAT in human serum by kinetic fluorescence assay The functions of INBRX-101 and endogenous AAT at 1.00 μM to 8.00 μM in human serum were verified using a kinetic fluorescence assay. This assay consists of quantifying the total functional concentrations of INBRX-101 and endogenous AAT in human serum. The functional AAT levels in serum samples are determined by their ability to inhibit the activity of neutrophil elastase (NE), which is measured as the change in the kinetic rate of NE enzyme activity on its substrate. Thus, the NE enzyme activity (assay response) is inverse to the AAT concentration, and higher AAT concentrations result in lower NE enzyme activity. Due to the nature of the functional assay and the presence of endogenous AAT in serum at measurable concentrations, this method was investigated for PD biomarker analysis of endogenous AAT activity in combination with INBRX-101 drug activity. The assay results were relatively quantitative and were reported in equivalent AAT activity units in μM. The calibration curve was generated from plasma-derived AAT (pdAAT) as a reference standard.

[0115] Furthermore, since endogenous AAT was found at concentrations near or above the assay ULOQ in the target test population, this assay used a surrogate matrix approach where calibrators and quality controls were prepared in AAT-depleted serum (strip matrix). A pool of AAT-depleted human serum (strip matrix, lot number BRH1592175 or equivalent) was used as the surrogate matrix to prepare standards, QCs, and dilute samples. Briefly, standards, QCs, and samples containing AAT were diluted to the assay MRD and then combined with neutrophil elastase (NE) in buffer. The mixture was incubated for 30 minutes to allow AAT to irreversibly inhibit NE. Next, the samples were added to a reading plate containing the AAPV substrate, and the enzyme reaction was read for 10 minutes. The resulting kinetic data were reduced and reported in units of Vmax (RFU / second). Using the standard AAT concentrations and their resulting Vmax data, a standard curve was regressed, and the concentration of equivalent AAT in the samples was determined against it.

[0116] Example 1 - Evaluation of the Safety and Pharmacokinetics of Recombinant Human AAT-Fc Fusion Protein INBRX-101 in Patients with α-1 Antitrypsin Deficiency A non-blind international Phase 1 trial was conducted to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of INBRX-101. AATD patients were administered single or multiple doses (three consecutive doses every three weeks) of 10, 40, 80, or 120 mg / kg INBRX-101 by intravenous infusion.

[0117] Table 1 shows the dose levels of the single ascending dose (SAD) and multiple ascending dose (MAD) of INBRX-101, the number of subjects in each dose level group, and the number of administrations.

[0118] No drug-related serious or severe adverse events were observed in 24 AATD patients at doses including a maximum single dose of 120 mg / kg and a multiple dose of 80 mg / kg. Drug-related adverse events (AEs) were mainly mild, with a few moderate events, all of which were transient and reversible. The most commonly reported drug-related adverse events were fatigue (n = 5), pruritus (n = 5), increased blood pressure (n = 5), urticaria (n = 4), and infusion-related reactions (n = 2). Infusion-related reactions (e.g., pruritus, increased blood pressure, urticaria, infusion-related reactions) were transient and mostly mild (Grade 1 according to CTCAE v5.0) except for one moderate event (Grade 2) and responded well to symptomatic treatment.

[0119] Single dose: Serum antigenicity PK and functional AAT levels were evaluated in 21 AATD patients. Over the dose range of 10 - 120 mg / kg above baseline (about 1 mM), a dose-related increase in maximum and total INBRX-101 exposure was observed. A slight increase in serum AAT trough was observed over a six-month period at single doses of 80 and 120 mg / Kg (see Figures 2A - 2B). A higher AAT trough was observed with minimal variability at a dose of 120 mg / ml, and 4 / 5 of the subjects tested showed a trough above 20 mM. An elimination half-life of approximately 15 - 19 days was calculated. Functional AAT levels increased rapidly after administration of INBRX-101 and showed a dose response with respect to maximum and 21-day concentrations (see Figure 1).

[0120] Multiple Doses: Each of the multiple-dose regimens administered every three weeks is one of 40, 80, or 120 mg / Kg. Data over a six-month period are disclosed herein. Preliminary data from multiple administrations of 40 mg / kg or 80 mg / kg every three weeks showed accumulation along with an extended half-life. Higher functional AAT levels above baseline and within the normal physiological range were observed at 84 days post-treatment. The observed maximum (Cmax) and trough levels of functional AAT caused by the multiple-dose regimens exceeded those historically reported for plasma-derived AAT and maintained AAT levels within the normal physiological levels (Figures 3A - 3B).

[0121] Additional data from 31 AATD patients in this Phase 1 trial (26 were of the ZZ genotype of the SERPINA1 gene, 3 were of the SZ genotype, and 2 were of the MZ genotype) showed that treatment was well tolerated without drug-related severe or serious adverse events. Drug-related adverse events were mainly mild, and all of the few adverse events of moderate severity were transient and reversible, with minimal or no symptomatic treatment. No safety-related or PK / PD-related signs of neutralizing anti-drug antibodies were observed.

[0122] Dose-related increases in maximum and total INBRX-101 exposure occurred across the entire single and multiple escalating dose ranges. Data from multiple escalating dose cohorts of INBRX-101 at 40, 80, and 120 mg / kg IV every three weeks showed the expected accumulation of functional AAT levels (Figure 4A). Based on PK modeling, the accumulation continues after subsequent administrations and is expected to reach steady state approximately 5 - 6 consecutive times after once-weekly dosing for three weeks.

[0123] Current standard treatment plasma-derived AAT administered once weekly at 60 mg / kg achieves a Cavg of functional AAT of 17.8 μM over the weekly dosing interval, as calculated from the steady-state area under the curve (“AUC”) values reported by Stock et al. BMC Clinical Pharmacology 2010, 10:13. INBRX-101 achieved an average Cavg of functional AAT of 40.4 μM over a 21-day dosing interval following the third 80 mg / kg dose. Bronchoalveolar lavage fluid (“BALF”) samples from two individuals in the multiple escalating dose cohorts of 80 mg / kg that have been processed to date confirm the presence of INBRX-101 in the lung fluid. Additionally, functional AAT levels were measured in plasma samples from 65 normal MM genotype individuals. This analysis revealed that the 5th percentile and 95th percentile of functional AAT levels in normal MM genotype individuals were 23 μM and 57 μM, respectively, with a median of 38 μM (Figure 4A).

[0124] The results described herein demonstrate that INBRX-101 is generally safe and well-tolerated and has the potential to maintain normal AAT serum levels above 20 μM over the entire dosing interval in a dosing schedule that may be administered once monthly, every three weeks. INBRX-101 achieved and maintained normal AAT serum levels above 20 μM at three-week intervals or potentially longer dosing intervals. The results disclosed herein also show that the disclosed INBRX-101 dosing regimen has the potential to reduce the total number of infusions in a year while maintaining the patient within the normal range of functional AAT.

[0125] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, are to be interpreted in an inclusive sense, that is as meaning “including”, but not as excluding any other integers, steps, integer groups or groups of steps.

[0126] As used herein, the term "about", unless otherwise indicated, refers to the recited value, e.g., amount, dosage, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0127] All patents, patent applications, and references cited throughout the specification of the present invention are hereby incorporated by reference in their entirety.

[0128] The present invention encompasses all combinations of preferred groups, more preferred groups, suitable groups, and embodiments of the groups listed above.

Claims

1. A method for treating or alleviating symptoms associated with abnormal serine protease activity in a subject in need of such a method, the method comprising administering to the subject an AAT-Fc fusion protein by infusion at an initial dose of about 10 to 120 mg / Kg on the first day of treatment and subsequent doses of 10 to 120 mg / Kg every 3 or 4 weeks thereafter, wherein the AAT-Fc fusion protein comprises (i) the amino acid sequence of SEQ ID NO: 1 or (ii) an AAT polypeptide of SEQ ID NO: 2 and an Fc polypeptide of SEQ ID NO:

3.

2. The method of claim 1, comprising administering an initial dose or subsequent dose of about 60 to 120 mg / Kg.

3. The method of claim 1, comprising administering an initial dose or subsequent dose of about 40 to 80 mg / Kg.

4. The method of claim 1, 2, or 3, comprising administering an initial dose or subsequent dose of about 80 mg / Kg.

5. The method of claim 1 or 2, comprising administering an initial dose or subsequent dose of about 120 mg / Kg.

6. The method according to any one of claims 1 to 5, wherein the subsequent dose is higher than the initial dose or a previous subsequent dose.

7. The method according to any one of claims 1 to 5, wherein the subsequent dose is lower than the initial dose or a previous subsequent dose.

8. The method according to any one of claims 1 to 5, wherein the subsequent dose is the same as the initial dose or a previous subsequent dose.

9. The method according to any one of claims 1, 2, or 5 to 8, comprising administering an initial dose of about 120 mg / Kg on the first day of treatment and subsequent doses every 3 weeks thereafter.

10. The method according to any one of claims 1, 2, or 5 to 8, comprising administering a first dose of about 120 mg / Kg on the first day of treatment, and subsequent doses every 4 weeks thereafter.

11. (a) Determining the level of expression or activity of a serine protease in a subject prior to administration of a first dose to obtain a baseline of expression or activity; (b) Determining the level of expression or activity of the serine protease in a period of at least 3 weeks after administration of the first dose or a subsequent dose; and (c) If the expression or activity of the serine protease in the subject is above the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is greater than or equal to the previous dose of the AAT-Fc fusion protein; or (d) If the expression or activity of the serine protease in the subject is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is lower than the previous dose The method according to any one of claims 1 to 3, further comprising.

12. (a) Determining the level of expression or activity of AAT in a subject prior to administration of a first dose to obtain a baseline of expression or activity; (b) Determining the level of expression or activity of AAT in a period of at least 3 weeks after administration of the first dose or a subsequent dose; and (c) If the expression or activity of AAT in the subject is below the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is greater than or equal to the previous dose of the AAT-Fc fusion protein; or (d) If the expression or activity of AAT in the subject is higher than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein that is lower than the previous dose The method according to any one of claims 1 to 3, further comprising.

13. (a) determining the serum AAT level in the subject during a period of at least 3 weeks after administration of the first or subsequent dose of the AAT-Fc fusion protein to obtain the serum AAT level; and (b) if the serum AAT level in the subject is below the normal range, administering a subsequent dose of the AAT-Fc fusion protein that is greater than or equal to the previous dose of the AAT-Fc fusion protein; or (c) if the serum AAT level in the subject is higher than the normal range, administering a subsequent dose of the AAT-Fc fusion protein that is lower than the previous dose The method according to any one of claims 1 to 3, further comprising.

14. The method according to claim 13, wherein the functional AAT level is determined.

15. The method according to claim 13 or 14, wherein the serum AAT level in the subject is less than about 15 μM or greater than about 50 μM.

16. The AAT-Fc fusion protein is an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 5 mM Tris; about 150 mM trehalose; about 100 mM sucrose; about 100 mM proline; about 2 mM methionine; and about 0.1% (w / v) poloxamer in an aqueous solution comprising; the pH of the aqueous solution is adjusted to about 7.3 using either hydrochloric acid or sodium hydroxide; the total ionic strength of the aqueous solution excluding the contribution of the AAT-Fc fusion protein is about 4.3 mM, The method according to any one of claims 1 to 15.

17. the AAT-Fc fusion protein is an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20 The method according to any one of claims 1 to 15, which is in an aqueous solution containing

18. The method according to claim 17, wherein the aqueous solution has a pH of about 7.

0.

19. The method according to any one of claims 16 to 18, wherein the aqueous solution contains about 50 mg / ml of the AAT-Fc fusion protein.

20. The method according to any one of claims 1 to 19, wherein the subject in need of the method has abnormal serine protease activity associated with a disease or disorder selected from the following: AAT deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing diseases or disorders, systemic lupus erythematosus and multiple sclerosis.

21. The method according to claim 20, wherein the subject has AAT deficiency.

22. The method according to claim 20, wherein the subject has an infectious disease selected from a bacterial infection, a fungal infection, or a viral infection.

23. The method according to any one of claims 1 to 22, wherein the subject is a human.

24. The method according to any one of claims 1 to 23, wherein the infusion is delivered over a period of about 30 to 120 minutes. **Claim 25** The method according to claim 24, wherein the infusion is delivered over a period of about 30 to 60 minutes. **Claim 26** The method according to any one of claims 1 to 25, wherein the subject in need of the method has a serum AAT level of less than 20 μM prior to the first dose. **Claim 27** The method according to any one of claims 1 to 26, wherein the subject in need of the method has a serum AAT level of 11 μM or less prior to the first dose. **Claim 28** A unit dose vial comprising: an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1 at about 5 mg / ml to about 100 mg / ml; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20 A unit dose vial comprising the same. **Claim 29** The unit dose vial according to claim 28, comprising about 50 mg / ml of the AAT-Fc fusion protein. **Claim 30** The unit dose vial according to any one of claims 28 or 29, wherein the pH is about 7.0.

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