Effective dose of recombinant serine protease inhibitor-Fc fusion protein for use in method of treating AAT deficiency in subject

Abstract

The present application relates to an aqueous solution comprising an engineered AAT-Fc fusion dimer protein in an effective dose for use in a method of treating or alleviating a symptom associated with aberrant serine protease activity in a subject in need thereof.

Description

Related Applications

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 342,264, filed on May 16, 2022, and U.S. Provisional Application No. 63 / 492,692, filed on March 28, 2023, the contents of which are hereby incorporated by reference in their entirety. Incorporation by Reference into the Sequence Listing

[0002] The sequence listing XML associated with this application is provided electronically in XML file format and is hereby incorporated into the specification by reference. The 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 is 4.90 kilobytes in size. Technical Field

[0003] The present invention relates to an aqueous solution containing human AAT-IgG Fc fusion protein in an effective dosage range and an administration interval of the AAT-IgG Fc fusion protein for treating AAT deficiency. Background Art

[0004] Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed genetic disease that affects about 100,000 patients in the United States. It is characterized by insufficient AAT levels, leading to emphysema, loss of lung function and shortened life expectancy. Based on biochemical efficacy, plasma-derived AAT (pdAAT) therapy was approved in the 1980s and is administered once a week to maintain serum AAT concentrations above 11 μM (below the normal range). In addition, it is reported that AAT therapy can regulate inflammation and is being studied for inhibiting inflammatory diseases and disorders. Since then, new treatment modalities have made little progress, and the cost / supply of pdAAT remains a challenge. A variety of engineered antibody proteins have been developed, including bispecific antibodies and trispecific antibodies. Many 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) can play a purpose different from its physiological purpose, particularly the purpose of extending 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 comprising serine protease inhibitor (serpin) polypeptides or amino acid sequences derived from serine protease inhibitors. It is necessary to determine the dosage regimens for these engineered proteins to be used for effective treatment and half-life extension in the subject's system. The present invention addresses this need. Summary of the invention

[0005] The present disclosure provides a method for 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 every three or four weeks thereafter, wherein the AAT-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or comprises an AAT polypeptide of SEQ ID NO: 2 and an Fc polypeptide of SEQ NO: 3.

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

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

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

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

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

[0011] In some embodiments, the method comprises 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 comprises 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 further comprises: (a) determining the level of serine protease expression or activity of the subject prior to administration of the first dose to obtain a baseline of expression or activity; (b) determining the level of serine protease expression or activity of the subject over a period of at least three weeks after administration of the first or subsequent dose; and (c) when the serine protease expression or activity of the subject is equal to or higher than 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), the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (d) when the serine protease expression or activity of the subject is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being lower than the previous dose.

[0014] In some embodiments, the method further comprises: (a) determining the level of AAT expression or activity of the subject before administering the first dose to obtain a baseline of expression or activity; (b) determining the level of AAT expression or activity of the subject for a period of at least three weeks after administering the dose; and (c) when the subject's AAT expression or activity is equal to or higher than 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), the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (d) when the subject's AAT expression or activity is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being lower than the previous dose.

[0015] In some embodiments, the method further comprises: (a) determining the serum AAT level of the subject for a period of at least three weeks after administering the first or subsequent dose of the AAT-Fc fusion protein to obtain a serum AAT level; and (b) administering a subsequent dose of the AAT-Fc fusion protein when the subject's serum AAT level is below the normal range, the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (c) administering a subsequent dose of the AAT-Fc fusion protein when the subject's serum AAT level is above the normal range, the subsequent dose being lower than the previous dose. In some embodiments of the above methods, a functional AAT level is determined. In some embodiments of the above methods, the subject's serum AAT level 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: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; 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 hydrochloric acid or sodium hydroxide; and wherein 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: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; 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 AAT-Fc fusion protein.

[0019] In some embodiments, the subject in need thereof has abnormal serine protease activity associated with a disease or condition selected from the group consisting of 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 following cardiac 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 a human.

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

[0023] The present disclosure also provides a unit dose vial comprising: about 5 mg / ml to about 100 mg / ml of an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; 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 wherein the pH is 7.0.

[0024] In some embodiments, a unit dose vial contains about 50 mg / ml of AAT-Fc fusion protein.

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

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, in particular, U.S. Provisional Application No. 63 / 342,264 filed on May 16, 2022 and U.S. Provisional Application No. 63 / 492,692 filed on March 28, 2023, are incorporated herein by reference in their entirety for any purpose. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples described herein are only illustrative and are not intended to be restrictive.

[0027] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Is a graph depicting the increase in functional AAT levels in serum of all subjects in each dose level group administered different single dose levels of INBRX-101 at baseline, the maximum AAT levels, and the AAT levels on day 21 after administration, as indicated. The shaded area of ​​the graph represents the normal physiological level of AAT in human serum. The y-axis represents the functional AAT level (μM), and the x-axis represents the individual INBRX-101 single dose level (mg / Kg).

[0029] Figure 2A-2B are two graphs depicting the dose-dependent increase in functional AAT levels following administration of INBRX-101. Figure 2A is a graph depicting the mean increase in functional AAT levels in serum of subjects in each dose level group administered different single dose levels of INBRX-101 at baseline, the maximum AAT level, and the AAT level on day 21 after administration. Figure 2B It is depicted in the detection Figure 2A Figure 2 is a graph of the average increase in the level of functional AAT in the serum of subjects in each dose level group of INBRX-101 at different single dose levels 6 months after administration of the functional AAT level in the serum of subjects in each dose level group. The shaded area of ​​the graph represents the normal physiological level of AAT in human serum. The y-axis represents the functional AAT level (μM), and the x-axis represents the individual INBRX-101 single dose level (mg / Kg).

[0030] Figure 3A-3B are two graphs depicting a comparison of the dose-dependent increase in functional AAT levels following administration of INBRX-101 or Zemaira. Figure 3Adepicts the maximum levels (Cmax) observed for each dose level of INBRX-101 and Zemaira at Dose 1, Dose 2, and steady-state (baseline), and Figure 3B Depicted are the trough values ​​of functional AAT levels at dose 1, dose 2, and steady state (baseline) for each dose level of INBRX-101 and Zemaira. The shaded area of ​​the graph represents the normal physiological level of AAT in human serum. The y-axis represents the functional AAT level (μM), and the x-axis represents the individual INBRX-101 single dose level (mg / Kg).

[0031] Figure 4A-4B are two graphs depicting functional AAT levels over time in AATD patients administered 40, 80, or 120 mg / kg INBRX-101 every three weeks. Figure 4A Depicted are functional AAT levels (μM) for patients who were administered multiple doses at intervals of 3 weeks at 40, 80, and 120 mg / Kg (as indicated). The shaded area represents the normal functional AAT range for healthy adults. The y-axis represents functional AAT levels (μM), and the x-axis represents individual INBRX-101 dose time points with arrows. Figure 4B Depicts the baseline levels (μM) of functional AAT in the plasma of 65 healthy volunteers and 30 patients in the Phase I AAT study before administration of INBRX-101. The y-axis represents the functional AAT level (μM), and the x-axis represents the identity of healthy volunteers and AAT variant patients (determined by Mayo Clinic Laboratories using LC-MS / MS method (A1ALC)). The box plot shows the minimum, lower quartile, median, upper quartile, and maximum. The shaded area represents the 5th to 95th percentile of the normal functional AAT range for adults with healthy MM genotypes. DETAILED DESCRIPTION

[0032] The present disclosure is based, in part, on the unexpected discovery that administration of an alpha-1 antitrypsin (AAT) Fc fusion protein (also referred to herein as INBRX-101) to individuals with alpha-1 antitrypsin deficiency (AATD) can restore alpha-1 antitrypsin to normal levels.

[0033] AATD is a genetic disease characterized by insufficient levels of AAT, leading to emphysema, loss of lung function, and shortened life expectancy. Current augmentation therapy with plasma-derived AAT requires weekly IV dosing due to its short half-life and is designed to maintain patients' target serum AAT concentrations above 11μM. In contrast, INBRX-101 is an engineered recombinant human AAT-Fc fusion protein that has been shown to achieve serum AAT levels within the normal range above 20μM over dosing intervals of at least three weeks. INBRX-101 is the first ATT-Fc fusion protein to achieve and maintain serum AAT levels within the normal range (>20μM) using extended dosing intervals.

[0034] The extended dosing interval of INBRX-101 can reduce the frequency of infusion, eliminate the lung decline caused by AATD and significantly improve the quality of life of patients. Therefore, the present disclosure provides a method for treating or alleviating the symptoms associated with abnormal serine protease activity in a subject, the method being performed as follows: administering an AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) to the subject by infusion at a dose of about 10 to 120 mg / Kg on the first day of treatment and every three or four weeks thereafter. In some embodiments, serum AAT levels (e.g., functional AAT levels) are generally maintained at> 20 μM during administration.

[0035] The amino acid sequence of the INBRX-101 monomer is shown below. The AAT polypeptide portion of INBRX-101 (SEQ ID NO: 2) is underlined, with the Met351Glu mutation in bold and italics, and the Met358Leu mutation in bold and italics: and the IgG4-Fc polypeptide portion of INBRX-101 (SEQ ID NO: 3) is in italics, with mutations S228P, L235E, M252Y and M428L shown in boxes. The GS linker connecting the AAT polypeptide portion and the IgG4-Fc polypeptide portion is in bold.

[0036] INBRX-101 is produced as a monomer, but can form dimers in aqueous solution. In some embodiments, INBRX-101 disclosed herein can exist as a mixture of monomeric and dimeric proteins. In some embodiments, the monomers of the dimeric protein can be connected to each other by disulfide bridges. In particular, human immunoglobulin Fc polypeptides or polypeptides derived from immunoglobulin Fc polypeptides are so connected to form a functional Fc domain.

[0037] 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 to 120 mg / Kg (e.g., about 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 110 to 115, or 115 to 120 mg / Kg). In some embodiments, the dose is between about 40 to 80 mg / Kg (e.g., about 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, or 75 to 80 mg / Kg). In some embodiments, the dosage is between about 60 and 120 mg / Kg (e.g., about 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 1110 to 115, or 115 to 120 mg / Kg). In some embodiments, the dosage is between about 60 and 80 mg / Kg (about 60 to 65, 65 to 70, 70 to 75, or 75 to 80 mg / Kg).

[0038] In some embodiments, the dosage is about 40 mg / Kg. In some embodiments, the dosage is about 80 mg / Kg. In some embodiments, the dosage is about 120 mg / Kg.

[0039] In some embodiments, a dose of about 80 mg / Kg is administered on the first day of treatment and every three weeks thereafter.

[0040] In some embodiments, a dose of about 80 mg / Kg is administered on the first day of treatment and every four weeks thereafter.

[0041] In some embodiments, a dose of about 120 mg / Kg is administered on the first day of treatment and every three weeks thereafter.

[0042] In some embodiments, a dose of about 120 mg / Kg is administered on the first day of treatment and every four weeks thereafter.

[0043] In some embodiments, the subsequent dose is higher than the previous dose. In some embodiments, the subsequent dose is lower than the previous dose. In other embodiments, the subsequent dose is the same as the previous dose.

[0044] In some embodiments, the time period between each dose is the same.

[0045] In some embodiments, each subsequent dose is administered three weeks after the previous dose.

[0046] In some embodiments, each subsequent dose is administered four weeks after the previous dose.

[0047] In some embodiments, one or more subsequent doses are administered 3 weeks after the previous dose, followed by administration of one or more additional doses administered four weeks after the previous dose.

[0048] The AAT-Fc fusion protein (eg, INBRX-101; SEQ ID NO: 1) is administered by infusion (ie, intravenously).

[0049] In certain embodiments, the AAT-Fc fusion protein is diluted into an infusion bag containing a suitable diluent (e.g., glucose in saline, water, etc.). Due to the possibility of infusion or allergic reactions, it is recommended that premedication prevent such infusion reactions, and during the administration of the antibody, attention should be paid to preventing allergic reactions. In certain embodiments, the infusion will be administered to the subject within a time period between about 30 minutes and about 4 hours. In certain embodiments, if the subject does not show signs or symptoms of adverse infusion reactions, the IV infusion is delivered within a time period of about 30-240 minutes, about 30-180 minutes, about 30-120 minutes, or about 30-90 minutes, or within a time period of about 30-60 minutes, or within a shorter time period. In one embodiment, the IV infusion is delivered within a time period of about 30-60 minutes. In another embodiment, the infusion is delivered within a time period of about 35-55 minutes. In another embodiment, the IV infusion is delivered within a time period of about 45 minutes.

[0050] Typically, in the above embodiments, administration occurs at a predetermined frequency or period, or within about 1-3 days of such a scheduled interval, such that administration occurs 1-3 days before the scheduled dose, 1-3 days after the scheduled dose, or on the day of the scheduled dose, for example once every 3 weeks (± 3 days).

[0051] In some embodiments, the method further comprises: (a) determining the level of serine protease expression or activity of the subject before administration of the first dose to obtain a baseline of expression or activity; (b) determining the level of serine protease expression or activity of the subject for a period of at least three weeks after administration of a dose in the method of the present disclosure; and (c) when the serine protease expression or activity of the subject is equal to or higher than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being equal to or higher than the previous dose of the serine protease fusion protein; or (d) when the serine protease expression or activity of the subject is lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being lower than the previous dose.

[0052] In some embodiments, the serine protease activity of the subject in step (a) of the methods disclosed herein is above the physiological level of serine protease activity in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0053] 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 the physiological level of serine protease activity in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0054] Symptoms of aberrant serine protease expression or activity (eg, decreased from normal AAT levels) may include, for example, shortness of breath, excessive coughing with phlegm / sputum production, wheezing, increased chest pain on inspiration, decreased exercise capacity, and persistent low energy state or fatigue.

[0055] In some embodiments, the serine protease activity of a subject can be determined by any conventional method for detecting protein / enzyme activity in a subject's tissue (e.g., kinetic fluorescence assays, spectrophotometric enzyme assays, calorimetric enzyme assays, light scattering enzyme assays, and microthermophoresis, etc.).

[0056] In some embodiments, the subject in step (a) of the methods disclosed herein expresses serine protease above physiological levels of serine protease activity in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0057] In some embodiments, the serine protease expression of the subject in step (a) of the method disclosed herein is at least about 1.5 to 2 times the physiological level of serine protease activity in normal subjects without any symptoms or disorders associated with abnormal serine protease expression or activity. The normal physiological level of exemplary serine protease neutrophil elastase (NE) in human plasma is about 32 to 56 μg / L. In some embodiments, in subjects with abnormal serine protease expression, the level of exemplary serine protease NE can be at least about 48 to 112 μg / L (e.g., about 48 to 52, 52 to 56, 56 to 60, 60 to 66, 66 to 70, 70 to 74, 74 to 78, 78 to 82, 82 to 86, 86 to 90, 90 to 96, 96 to 100, 100 to 104, 104 to 108 or 108 to 112 μg / L).

[0058] In some embodiments, serine protease expression in a subject can be determined by any conventional method for detecting protein / enzyme expression in a subject's tissue (e.g., electrochemiluminescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, mass spectrophotometry, etc.).

[0059] Serine proteases are catalytic enzymes produced by the liver in response to pathological conditions (such as infection). Alpha-antitrypsin (AAT) is a serine protease inhibitor that regulates the activity of serine proteases (e.g., neutrophil elastase (NE)). Therefore, the level of serine protease activity is inversely proportional to the level of AAT expression and activity. Subjects with a physiological level of serine protease activity higher than normal subjects (without any symptoms or illness associated with abnormal serine protease expression or activity) can also have an AAT lower than the physiological level of serine protease activity in normal subjects (without any symptoms or illness associated with abnormal serine protease expression or activity).

[0060] In some embodiments, the method further comprises: (a) determining the level of AAT expression or activity of the subject before administration of the first dose to obtain a baseline of expression or activity; (b) determining the level of AAT expression or activity of the subject for a period of at least three weeks after administration of the dose in the method of the present disclosure to obtain a baseline level of AAT expression or activity; and (c) when the subject's AAT expression or activity is equal to or lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (d) when the subject's AAT expression or activity is higher than 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), the subsequent dose being lower than the previous dose.

[0061] In some embodiments, the method further comprises: (a) determining the serum AAT level of the subject for a period of at least three weeks after administering the first or subsequent dose of the AAT-Fc fusion protein to obtain a serum AAT level; and (b) administering a subsequent dose of the AAT-Fc fusion protein when the subject's serum AAT level is below the normal range, the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (c) administering a subsequent dose of the AAT-Fc fusion protein when the subject's serum AAT level is above the normal range, the subsequent dose being lower than the previous dose. In some embodiments of the above methods, a functional AAT level is determined. In some embodiments of the above methods, the subject's serum AAT level is less than 15 μM or greater than 50 μM.

[0062] In some embodiments, the AAT activity of the subject in step (a) of the methods disclosed herein is below the physiological level of AAT activity in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0063] In some embodiments, the AAT activity of the subject in step (a) of the methods disclosed herein is at least about 1.5 to 2 times the physiological level of AAT activity in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0064] In some embodiments, the subject in step (a) of the methods disclosed herein expresses AAT below the physiological level of AAT expression in a normal subject without any symptoms or disorders associated with aberrant serine protease expression or activity.

[0065] 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 of the physiological level of AAT expression in a normal subject without any symptoms or disorders associated with abnormal serine protease expression or activity. The normal physiological level of AAT in human plasma is about 20 to 48 μM (80 to 220 mg / dL). In some embodiments, the AAT level in the subject's plasma is about 10 to 32 μM (e.g., about 10 to 12, 12 to 14, 14 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24, 24 to 26, 26 to 28, 28 to 30, 30 to 32 μM). AAT deficiency (AATD) is associated with a plasma concentration of less than 20 μM (e.g., 19 to 20, 18 to 19, 17 to 18, 16 to 17, 15 to 16, 14 to 15, 13 to 14, 12 to 13, 11 to 12, or <11 μM). In some embodiments, if the subject's plasma level is demonstrated to be ≤11 μM, the subject has severe AAT deficiency.

[0066] In some embodiments, the subject's AAT activity can be determined by any conventional method for detecting protein / enzyme activity in the subject's tissue (e.g., kinetic fluorescence assay, spectrophotometric enzyme assay, calorimetric enzyme assay, light scattering enzyme assay, human anti-neutrophil elastase capacity (ANEC) assay, and microthermophoresis, etc.) (see, e.g., Engelmaier A, Weber A. (2022) JPharm Biomed Anal. [Journal of Pharmacy and Biomedical Analysis]; 209: 114476).

[0067] In some embodiments, AAT expression in a subject can be determined by any conventional method for detecting protein / enzyme expression in a tissue of a subject (e.g., electrochemiluminescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, mass spectrophotometry, etc.).

[0068] In some embodiments, the expression / activity of AAT or serine proteases or both can be measured in tissues and body fluids (including blood, serum, plasma, sputum, urine, feces, bronchoalveolar lavage fluid, vaginal lavage fluid, semen, etc.) of a subject.

[0069] In some embodiments, wherein a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than a previous dose of the AAT-Fc fusion protein, the first or initial dose of the AAT-Fc fusion protein is about 40 to 80 mg / Kg (e.g., 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, or 75 to 80 mg / Kg), and the subsequent dose of the AAT-Fc fusion protein is about 80 to 120 mg / kg (e.g., 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 110 to 115, or 115 to 120 mg / Kg). In some embodiments, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than the 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, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than the 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.

[0070] In some embodiments, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than the 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, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than the 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, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is equal to or higher than the 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.

[0071] In some embodiments, wherein a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is lower 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 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 110 to 115, or 115 to 120 mg / Kg), and the subsequent dose of the AAT-Fc fusion protein is about 40 to about 80 mg / kg (e.g., 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, or 75 to 80 mg / Kg).

[0072] In some embodiments, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is lower than the 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 40 mg / kg. In some embodiments, wherein the subsequent dose of the AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is lower than the 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 80 mg / Kg. In some embodiments, wherein a subsequent dose of an AAT-Fc fusion protein (e.g., INBRX-101: SEQ ID NO: 1) is lower 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 40 mg / Kg.

[0073] In some embodiments, subsequent doses are administered every three 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 week, 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 week, 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 week, every 42 weeks, every 43 weeks, every 44 weeks, every 45 weeks, every 46 weeks, every 47 weeks, or every 48 weeks (or 12 months) after the first or prior dose.

[0074] In some embodiments, subsequent doses are administered every three weeks after a first or previous dose. In some embodiments, subsequent doses are administered every four weeks after a first or previous dose.

[0075] In some embodiments, the subject in need thereof has abnormal serine protease activity associated with a disease or condition selected from the group consisting of 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 following cardiac 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.

[0076] In some embodiments, the subject suffers from an infection selected from a bacterial infection, a fungal infection, or a viral infection. In some embodiments, the subject is a mammal. In some embodiments of the use of any method according to the present disclosure or according to the purposes of any method of the present disclosure, the subject is a human, a rodent, a feline, a dog, a cow, a horse, a camel, or a mammalian subject. In some embodiments of the use of any method according to the present disclosure or according to the purposes of any method of the present disclosure, the subject is a human.

[0077] Pharmaceutical composition

[0078] The engineered AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) of the present invention can be further incorporated into a pharmaceutical composition suitable for administration. Such a composition typically comprises 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, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference in this field and 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. Liposomes and non-aqueous vehicles (e.g., fixed oils) may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated.Supplementary active compounds can also be incorporated into the compositions.

[0079] The pharmaceutical composition of the present invention is formulated to be compatible with the 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 applications may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. pH can be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0080] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, The composition may be prepared by a pre-treatment of the composition with a phosphate buffered saline (PBS) or a pre-treatment of the composition with a CrEL (CrEL) (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should have fluidity to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must prevent the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity may be maintained, for example, by the use of a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by the use of a surfactant. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents (e.g., sugars), polyols (e.g., mannitol, sorbitol), and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0081] Sterile injectable solutions can be prepared by incorporating the AAT-fusion protein (e.g., INBRX-101; SEQ ID NO: 1) in the desired amount in an appropriate solvent together with one or a combination of the ingredients listed herein, followed by sterilization filtration as required. Dispersions are generally prepared by incorporating the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, which methods produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof.

[0082] In some embodiments, the pharmaceutical composition comprises a unit dose vial. In some embodiments, the unit dose vial comprises: about 5 mg / ml to about 100 mg / ml of an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.01% (w / v) polysorbate 20.

[0083] In some embodiments, the unit dose vial contains about 5 mg / ml to about 100 mg / ml of an AAT-Fc fusion protein (i.e., INBRX-101; SEQ ID NO: 1); about 9.5 mg / ml disodium phosphate heptahydrate; about 0.2 mg / ml monosodium phosphate monohydrate; about 7.26 mg / ml sodium chloride; about 20.0 mg / ml trehalose dihydrate; and about 0.1 mg / ml polysorbate 20 solution. In some embodiments, the unit dose vial contains: about 5 mg / ml to about 100 mg / ml of an AAT-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 1; about 9.5 mg / ml disodium phosphate heptahydrate; about 2.0 mg / ml monosodium phosphate monohydrate; about 7.26 mg / ml sodium chloride; about 20.0 mg / ml trehalose dihydrate; and about 0.1 mg / ml polysorbate 20 solution. In some embodiments, the unit dose vial contains about 50 mg / ml of AAT-Fc fusion protein. In some embodiments, the contents of the unit dose vial have a pH of about 7.0.

[0084] The pharmaceutical composition comprises an AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) in an aqueous solution at a pH ranging from 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; an uncharged tonicity modifier at 50 to 200 mM each; a surfactant at about 0.01 to 2 mg / ml; and optionally one or more neutral amino acids at 0 to 300 mM each.

[0085] The pKa of the aqueous solution is in the range of 4.0 to 10.0, and the pKa is within 2 pH units of the pH of the aqueous solution.

[0086] Excluding the contribution of the AAT-Fc fusion protein (eg, INBRX-101; SEQ ID NO: 1), the total ionic strength of the aqueous solution was less than 30 mM.

[0087] One or more 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-iminodiacetic acid, 2-[(2-amino)-1,2-diamino] ... -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 salts thereof, disodium phosphate heptahydrate and monosodium phosphate monohydrate or a combination thereof.

[0088] Exemplary uncharged tonicity modifiers include polyols, sugars (e.g., monosaccharides and disaccharides) and sugar alcohols. In some embodiments, the uncharged tonicity modifier is selected from the group consisting of glycerol, 1,2-propylene glycol, mannitol, sorbitol, glucose, sucrose, trehalose, PEG300 and PEG400.

[0089] The total concentration of the uncharged tonicity adjuster or the combination of more than one tonicity adjusters is 50-1000 mM, such as 200-600 mM, 200-500 mM, or wherein the total concentration of the uncharged tonicity adjuster or the combination of more than one 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 uncharged tonicity adjuster or the combination of more than one tonicity adjusters is 50-150 mM.

[0090] In some embodiments, the aqueous solution comprises one or more neutral amino acids selected from the group consisting of glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine.

[0091] 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, excluding the contribution of the AAT-Fc fusion protein, the total ionic strength of the aqueous solution is less than 20 mM.

[0092] As used herein, neutral amino acids are amino acids whose side chains do not contain ionizable groups that are significantly ionized at the pH of aqueous solution (e.g., more than 20%, especially more than 50% of the side chains carry 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, and particularly their L isomers.

[0093] In certain embodiments, the aqueous solution comprises a nonionic surfactant. In certain embodiments, the nonionic surfactant is selected from the group consisting of: alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers (poloxamers) of polyethylene glycol and polypropylene glycol, and alkylphenyl ethers of polyethylene glycol. In certain embodiments, the nonionic surfactant is a polysorbate, such as polysorbate 20 or polysorbate 80. In certain embodiments, the nonionic surfactant is a block copolymer (poloxamers) of polyethylene glycol and polypropylene glycol, such as poloxamer 188.

[0094] 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.

[0095] In some embodiments, the pH of the aqueous solution is between about 7.2 and about 7.5 (eg, 7.2, 7.3, 7.4, or 7.5).

[0096] In certain embodiments, the aqueous solution comprises a nonionic surfactant. In certain embodiments, exemplary nonionic surfactants include alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers (poloxamers) of polyethylene glycol and polypropylene glycol and alkylphenyl ethers of polyethylene glycol. In certain embodiments, exemplary nonionic surfactants include polysorbates, such as polysorbate 20 or polysorbate 80. In certain embodiments, exemplary nonionic surfactants include block copolymers (poloxamers) of polyethylene glycol and polypropylene glycol, such as poloxamer 188. In certain embodiments, nonionic surfactants are present in a concentration of 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).

[0097] Polysorbate 20 is a monoester formed from lauric acid and polyoxyethylene (20) sorbitan, wherein 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, wherein the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 20 is known under a range of trade names, including in particular Tween 20 and Alkest TW 20. Polysorbate 80 is known under a range of trade names, including in particular Tween 80 and Alkest TW 80. Other suitable polysorbates include polysorbate 40 and polysorbate 60.

[0098] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 5 mg / ml to about 100 mg / ml of an AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1, 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, wherein the pH of the aqueous solution is adjusted to about 7.3 using hydrochloric acid or sodium hydroxide; and wherein 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.

[0099] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; about 0.6 mg / ml Tris; about 51.3 mg / ml trehalose; about 34.23 mg / ml sucrose; about 11.5 mg / ml proline; about 0.3 mg / ml methionine; and about 1 mg / ml (0.1% (w / v)) poloxamer; wherein the pH of the aqueous solution is adjusted to about 7.3 using hydrochloric acid or sodium hydroxide; and wherein 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.

[0100] In some embodiments, the AAT-Fc fusion protein (e.g., INBRX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; about 0.54 mg / ml Tris base; about 0.716 mg / ml Tris hydrochloride 21; about 56.7 mg / ml trehalose dihydrate; about 34.2 mg / ml sucrose; about 11.5 mg / ml proline; about 0.3 mg / ml methionine; and about 1 mg / ml (0.1% (w / v)) poloxamer; wherein the pH of the aqueous solution is adjusted to about 7.3 using hydrochloric acid or sodium hydroxide; and wherein 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.

[0101] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; one or more buffers of a substance at 1 to 40 mM each; one or more tonicity modifiers at 50 to 100 mM each; about 1 mg / ml of a surfactant; and a solvent. 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: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; about 50 mM sodium phosphate; about 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and about 0.1 mg / ml polysorbate 20. 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] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 35.4 mM disodium phosphate heptahydrate, about 1.7 mM monosodium phosphate monohydrate, about 124.2 mM sodium chloride, about 52.9 mM trehalose dihydrate, and about 0.1 mg / ml polysorbate 20. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0104] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 35.4 mM disodium phosphate heptahydrate, about 15.6 mM monosodium phosphate monohydrate, about 124.3 mM sodium chloride, about 52.9 mM trehalose dihydrate, and about 0.01% (w / v) polysorbate 20. In some embodiments, the aqueous solution comprises about 50 mg / ml of the AAT-Fc fusion protein.

[0105] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution comprising: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 1; about 9.5 mg / ml disodium phosphate heptahydrate; about 0.2 mg / ml monosodium phosphate monohydrate; about 7.26 mg / ml sodium chloride; about 20.0 mg / ml trehalose dihydrate; and about 0.1 mg / ml 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.

[0106] In some embodiments, the AAT-Fc fusion protein is in an aqueous solution, the aqueous solution comprising: about 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 1; about 9.5 mg / ml disodium phosphate heptahydrate; about 2.0 mg / ml monosodium phosphate monohydrate; about 7.26 mg / ml sodium chloride; about 20.0 mg / ml trehalose dihydrate; and about 0.1 mg / ml 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.

[0107] It should be noted 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, DRLide). If desired, the pKa values ​​of the amino acid side chains present in a polypeptide can be estimated using a suitable calculator.

[0108] In some embodiments, the AAT-Fc fusion protein (e.g., INHBX-101; SEQ ID NO: 1) is in an aqueous solution that comprises a physiologically acceptable osmotic pressure and is therefore suitable for parenteral administration. Thus, the osmotic pressure of the aqueous solution is suitably 200-500 mOsm / L, e.g., about 300 mOsm / L. The aqueous solution is, for example, isotonic with human plasma. In another embodiment, the osmotic pressure of the aqueous solution is 300-500 mOsm / L, e.g., about 400-460 mOsm / L. The aqueous solution may also be hypotonic or hypertonic, such as those intended to be diluted prior to administration.

[0109] In certain embodiments, the aqueous solution may additionally include a preservative, such as a phenolic preservative or a benzyl preservative. Exemplary preservatives are suitably selected from the group consisting of phenol, metacresol, chlorocresol, benzyl alcohol, propylparaben and methylparaben, particularly phenol, metacresol and benzyl alcohol. The concentration of the preservative is typically 10-100mM, such as 20-80mM, such as 25-50mM. The optimal concentration of the preservative in the aqueous solution is selected to ensure that the aqueous solution passes the Pharmacopoeia Antimicrobial Effectiveness Test (USP <51> , Volume 32).

[0110] In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Exemplary biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The method for preparing such a formulation is obvious to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal 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.

[0111] It is particularly advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the art in compounding such active compounds for treating individuals.

[0112] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0113] The present invention will be further illustrated in the following examples, which should not limit the scope of the invention described in the claims.

[0114] Table 1. Dose levels of INBRX-101, the number of subjects in each dose level group, and the number of doses for single administered dose (SAD) and multiple administered dose (MAD). Other embodiments

[0115] Although the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims. Examples

[0116] General Methods

[0117] Detection and Quantification of Recombinant Human AAT-Fc Fusion Protein INBRX-101

[0118] An electrochemiluminescence (ECL) assay was used to detect and quantify INBRX-101 in human serum, where anti-alpha-1 antitrypsin (anti-AAT CX2115, InhibRx, #2019011) was coated onto an MSD high binding plate (ECL capable). INBRX-101 in standards, QCs, controls, and samples was captured onto the coated plate. After thorough washing of the wells to remove unbound compounds, ruthenium 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 then MSD reading buffer was added. The assay plate was then read using an MSD ECL plate reader. The electrochemiluminescent signal generated is relative to the amount of INBRX-101 present in the standards, quality control samples (QCs), controls, and test samples. The concentration of INBRX-101 was back-calculated based on nonlinear regression of the standards. Data were acquired using a Meso Scale Discovery (MSD) Sector S 600. Data acquisition and analysis were performed in SoftMax Pro software (version 5.2 or 7.0.1), SoftMax Pro protocol TM2381.00.

[0119] All calculated values ​​(including but not limited to mean concentration, SD, %CV and %RE) are reported to up to three significant figures. Overall statistical calculations (SD, %CV and %RE) were performed using concentration values ​​rounded to three significant figures.

[0120] The function of INBRX-101 and endogenous AAT in human serum was quantitatively determined by a kinetic fluorescence assay.

[0121] The function of INBRX-101 and endogenous AAT between 1.00 μM and 8.00 μM in human serum was verified using a kinetic fluorometric assay. The assay includes quantifying the total functional concentration of INBRX-101 and endogenous AAT in human serum. The functional AAT level in serum samples is determined by its ability to inhibit NE activity, measured as a change in the kinetic rate of NE enzyme activity to its substrate. Therefore, NE enzyme activity (assay reaction) is opposite to AAT concentration, where higher AAT concentrations lead to lower NE enzyme activity. Due to the nature of the functional assay and the presence of endogenous AAT at measurable concentrations in serum, this method is considered for PD biomarker analysis of endogenous AAT activity combined with INBRX-101 drug activity. The assay results are relatively quantitative and reported in μM units of equivalent AAT activity. A calibration curve is generated from plasma-derived AAT (pdAAT) as a reference standard.

[0122] In addition, since the concentration of endogenous AAT was found to be close to or above the assay ULOQ in the targeted study population, the assay uses an alternative matrix method, in which calibrators and quality controls are prepared in AAT-depleted serum (stripped matrix). A human serum pool (stripped matrix, batch number BRH1592175 or equivalent) depleted by AAT is used as an alternative matrix to prepare standards, QCs and dilute samples. In short, standards, QCs and samples containing AAT are diluted to the assay MRD and then combined with neutrophil elastase (NE) in a buffer. The mixture is incubated for 30 minutes to allow AAT to irreversibly inhibit NE. Next, the sample is added to a reading plate with an AAPV substrate and the enzymatic reaction is read for 10 minutes. The resulting kinetic data is simplified and reported in units of Vmax (RFU / second). The AAT concentration of the standard and the resulting Vmax data are used to regress the standard curve, according to which the concentration of equivalent AAT in the sample is determined.

[0123] Example 1 - Recombinant human AAT-Fc fusion protein INBRX-101 in patients with alpha-1 antitrypsin deficiency Safety and pharmacokinetic evaluation of

[0124] An open-label, international Phase 1 trial designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of INBRX-101 was conducted. AATD patients were administered a single dose or multiple doses (three consecutive doses every three weeks) of 10, 40, 80, or 120 mg / kg INBRX-101 by IV infusion.

[0125] Table 1 shows the dose levels of INBRX-101, the number of subjects in each dose level group, and the number of doses for single administration dose (SAD) and multiple administration dose (MAD).

[0126] In 24 patients with AATD, no drug-related severe or serious adverse events were observed at doses up to and including a single dose of 120 mg / kg and multiple doses of 80 mg / kg. Drug-related adverse events (AEs) were mainly mild events, a few were moderate events, and all were transient and reversible. The most common drug-related adverse events reported 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 most were mild (grade 1 according to CTCAEv5.0) except for one moderate event (grade 2), and responded well to symptomatic therapy.

[0127] Single doseSerum antigenic PK and functional AAT levels were evaluated in 21 AATD patients. Dose-related increases in INBRX-101 maximum and total exposures from baseline (approximately 1 mM) were observed over the 10 to 120 mg / kg dose range. Slight increases in serum AAT trough values ​​were observed over a 6-month period at single doses of 80 and 120 mg / kg (see Figure 2A-2B ). Higher AAT trough values ​​were observed at the 120 mg / ml dose, with minimal variability, showing trough values ​​> 20 mM in 4 / 5 subjects tested. The terminal half-life was calculated to be approximately 15-19 days. Functional AAT levels increased rapidly after administration of INBRX-101 and showed a dose response relative to the maximum and 21-day post-dose concentrations (see Figure 1 ).

[0128] Multiple doses: Multiple dose regimen, wherein each dose in the multiple dose regimen (being any one of 40, 80 or 120 mg / Kg) is administered once every 3 weeks. Data within a six-month period are disclosed herein. Preliminary data of multiple doses of 40 mg / kg or 80 mg / kg every three weeks show that accumulation is consistent with the extension of half-life. Functional AAT levels were observed to be higher than baseline 84 days after treatment and within the normal physiological range. The maximum levels (Cmax) and trough levels of functional AAT observed to be caused by multiple doses exceed those levels of plasma-derived AAT reported in history, and AAT levels are maintained within normal physiological levels ( Figure 3A-3B ).

[0129] Further data from 31 AATD patients (26 with ZZ genotype of SERPINA1 gene, 3 with SZ genotype of SERPINA1 gene, and 2 with MZ genotype of SERPINA1 gene) in the Phase 1 study showed that the treatment was well tolerated, with no severe or serious adverse events related to the drug. Drug-related adverse events were mainly mild, and a few of those with moderate severity were transient and reversible, requiring minimal or no symptomatic care. No safety-related or PK / PD-related signs of neutralizing anti-drug antibodies were observed.

[0130] INBRX-101 maximum and total exposure increased in a dose-related manner across the single and multiple ascending dose ranges. Data from multiple ascending dose cohorts of INBRX-101 at 40, 80, and 120 mg / kg IV every three weeks demonstrated the expected accumulation of functional AAT levels ( Figure 4A Based on PK modeling, accumulation is expected to continue after subsequent doses and reach steady-state after approximately 5 to 6 consecutive doses every three weeks.

[0131] The current standard of care (plasma-derived AAT, dosed once weekly at 60 mg / kg) achieves a Cavg of 17.8 μM functional AAT within a weekly dosing interval, calculated based on the steady-state area under the curve (“AUC”) values ​​reported in Stocks et al. BMC Clinical Pharmacology 2010, 10:13. INBRX-101 achieved a mean Cavg of 40.4 μM functional AAT within a 21-day dosing interval following the third 80 mg / kg dose. To date, bronchoalveolar lavage fluid (“BALF”) samples from two individuals in the 80 mg / kg multiple ascending dose cohort have been processed to confirm the presence of INBRX-101 in the lung fluid. In addition, functional AAT levels were measured in plasma samples from 65 individuals with normal MM genotypes. This analysis revealed that the 5th and 95th percentiles of functional AAT levels in individuals with normal MM genotypes were 23 and 57 μM, respectively, with a median of 38 μM ( Figure 4A ).

[0132] The results described herein show that INBRX-101 is generally safe and well tolerated, and has shown the potential to maintain normal AAT serum levels (>20 μM) throughout the dosing interval at a once-every-three-week dosing schedule (potentially monthly dosing). INBRX-101 achieved and maintained normal AAT serum levels above 20 μM at dosing intervals of every three weeks or potentially longer. The results disclosed herein also show that the disclosed dosing schedule of INBRX-101 has the potential to maintain patients within the normal functional AAT range while reducing the total number of infusions per year.

[0133] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprises" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of stated integers, steps, groups of integers or groups of steps but not the exclusion of any other integers, steps, groups of integers or groups of steps.

[0134] As used herein, unless otherwise indicated, the term "about" refers to ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a recited value (e.g., amount, dosage, temperature, time, percentage, etc.).

[0135] All patents, patent applications, and references mentioned throughout the present specification are incorporated herein by reference in their entirety.

[0136] The present invention includes all combinations of preferred and more preferred groups and suitable and more 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 thereof, the method comprising: administering the AAT-Fc fusion protein to the subject by infusion at a first dose of about 10 to 120 mg / Kg on the first day of treatment and subsequent doses of 10 to 120 mg / Kg every three or four weeks thereafter, The AAT-Fc fusion protein (i) comprises the amino acid sequence of SEQ ID NO: 1 or (ii) comprises the AAT polypeptide of SEQ ID NO: 2 and the Fc polypeptide of SEQ ID NO:

3.

2. The method of claim 1 comprising administering a first or subsequent dose of about 60 to 120 mg / Kg.

3. The method of claim 1 comprising administering a first or subsequent dose of about 40 to 80 mg / Kg.

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

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

6. The method of any one of claims 1-5, wherein the subsequent dose is higher than the first dose or the previous subsequent dose.

7. The method of any one of claims 1-5, wherein the subsequent dose is lower than the first dose or the previous subsequent dose.

8. The method of any one of claims 1-5, wherein the subsequent dose is the same as the first dose or the previous subsequent dose.

9. The method of any one of claims 1, 2, or 5-8, wherein the method comprises administering a first dose of about 120 mg / Kg on the first day of treatment and administering subsequent doses every three weeks thereafter.

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

11. The method according to any one of claims 1 to 3, further comprising: (a) determining the level of serine protease expression or activity in the 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 over a period of at least three weeks after administration of the first or subsequent doses; as well as (c) administering a subsequent dose of the AAT-Fc fusion protein when the subject's serine protease expression or activity is equal to or higher than the baseline level obtained in step (a), the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (d) administering a subsequent dose of the AAT-Fc fusion protein when the serine protease expression or activity of the subject is below the baseline level obtained in step (a), the subsequent dose being lower than the previous dose.

12. The method according to any one of claims 1 to 3, further comprising: (a) determining the level of AAT expression or activity in the subject prior to administration of the first dose to obtain a baseline of expression or activity; (b) determining the level of AAT expression or activity over a period of at least three weeks after administration of the first or subsequent doses; as well as (c) when the subject's AAT expression or activity is equal to or lower than the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being equal to or higher than the previous dose of the AAT-Fc fusion protein; or (d) when the subject's AAT expression or activity is above the baseline level obtained in step (a), administering a subsequent dose of the AAT-Fc fusion protein, the subsequent dose being lower than the previous dose.

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

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

15. The method of claim 13 or 14, wherein the subject's serum AAT level is below about 15 μM or above about 50 μM.

16. The method of any one of claims 1-15, wherein the AAT-Fc fusion protein is in an aqueous solution comprising: About 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 1; About 5mM 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 hydrochloric acid or sodium hydroxide; Excluding the contribution of the AAT-Fc fusion protein, the total ionic strength of the aqueous solution was about 4.3 mM.

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

18. The method of claim 17, wherein the pH of the aqueous solution is about 7.

0.

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

20. The method of any one of claims 1-19, wherein the subject in need thereof has abnormal serine protease activity associated with a disease or condition selected from the group consisting of 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 following cardiac 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 of claim 20, wherein the subject suffers from AAT deficiency.

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

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

24. The method of any one of claims 1-23, wherein the infusion is delivered over a period of about 30-120 minutes.

25. The method of claim 24, wherein the infusion is delivered over a period of about 30-60 minutes.

26. The method of any one of claims 1-25, wherein the subject in need thereof has a serum AAT level of less than 20 μM prior to the first dose.

27. The method of any one of claims 1-26, wherein the subject in need thereof has a serum AAT level prior to the first dose of less than or equal to 11 μM.

28. A unit dose vial comprising: About 5 mg / ml to about 100 mg / ml of the AAT-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 1; About 50 mM sodium phosphate; About 125 mM sodium chloride; about 2% (w / v) trehalose dihydrate; and About 0.01% (w / v) polysorbate 20.

29. The unit dose vial of claim 28, comprising about 50 mg / ml of the AAT-Fc fusion protein.

30. The unit dose vial of any one of claims 28 or 29, wherein the pH is about 7.0.

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