Prevention and treatment methods for cardiac dysfunction and COVID-19 caused by activin A antagonists

Abstract

The present invention provides methods for preventing and treating cardiac dysfunction, including cardiomyopathy and heart failure. The methods of the present invention feature the administration of a therapeutically effective amount of an activin A antagonist, e.g., an antibody that binds to human activin A and reduces or neutralizes its activity. The methods of the present invention are useful in preventing and treating cardiac disease due to a variety of causes, including viral diseases, e.g., COVID-19.

Description

[Technical Field]

[0001] (Reference to the sequence list) This application incorporates, by reference, a sequence listing prepared on August 19, 2021, and submitted in computer-readable format as file 10771WO01-Sequence, containing 87,383 bytes.

[0002] (Field of invention) The present invention belongs to the field of pharmaceuticals and relates to a method and pharmaceutical composition for preventing and treating cardiac dysfunction using an activin A antagonist comprising an anti-activin A antibody and its antigen-binding fragment, or a combination of such antibody or antigen-binding fragment with a myostatin inhibitor. [Background technology]

[0003] Activins belong to the transforming growth factor-beta (TGF-β) superfamily and exert a wide range of biological effects on cell proliferation, differentiation, and apoptosis. Activins are homodimers or heterodimers of inhibin βA, inhibin βB, inhibin βC, and inhibin βE, and different combinations of these create various members of the activin protein family. For example, activin A is a homodimer of inhibin βA, activin B is a homodimer of inhibin βB, while activin AB is a heterodimer of inhibin βA and inhibin βB, and activin AC is a heterodimer of inhibin βA and inhibin βC (Tsuchida, K. et al., Cell Commun Signal 7:15 (2009)).

[0004] Activin A binds to and activates receptor complexes on the cell surface known as activin type II receptors (types IIA and IIB, also known as ActRIIA and ActRIIB, respectively). Activation of these receptors leads to phosphorylation of activin type I receptors (e.g., Alk4 or 7), which in turn leads to phosphorylation of SMAD2 and SMAD3 proteins, formation of SMAD complexes (with SMAD4), and translocation of the SMAD complexes into the cell nucleus, where SMAD2 and SMAD3 function to regulate the transcription of various genes (Sozzani, S. and Musso, T., Blood 117(19):5013-5015 (2011)).

[0005] Activin A or other ligands (including GDF8 (myostatin), activin B, activin AB, inhibin A, inhibin B, GDF3, GDF11, Nodal, BMP2, BMP4, BMP7, BMP9, and BMP10) that bind to and activate ActRIIB are associated with a variety of symptoms, including muscle wasting in aging and disease, as well as pulmonary and cardiac symptoms. For example, overexpression of activin A in the mouse airway has been suggested to be involved in pulmonary pathologies such as acute lung injury and acute respiratory distress syndrome, which is attenuated via neutralization of activin A by a fusion protein consisting of the extracellular portion of the activin type II receptor ActRIIB fused to the Fc portion of human IgG1 (Apostolou et al., Am J Respir Crit Care Med., 185(4):382-391). Similarly, activin type II receptor (ActRII) ligands have been suggested to be involved in cardiac aging and heart failure. Inhibition of the ActRII pathway by an antibody that blocks ActRIIA and ActRIIB (CDD866), or by an ActRIIB-Fc fusion protein (RAP-031) that blocks pathway activation by binding to a circulating ActRII ligand, reduces cardiac ActRII signaling while restoring or preserving cardiac function (Roh et al., Sci. Transl. Med, 11, eaau8680, 2019).

[0006] Drugs that bind to multiple ActRII ligands or inhibit ActRII signaling in a broad sense are known to cause various adverse effects when administered to human patients. The precise roles of many ActRII ligands are still not fully understood, and there is a need for specific inhibitors of ActRII ligands that can provide clinical benefits. [Overview of the project]

[0007] In one embodiment, the present invention provides a method for preventing or treating cardiac dysfunction or heart failure in a subject in need thereof, the method comprising administering an activin A-specific antagonist to the subject.

[0008] In some embodiments, the activin A-specific antagonist is an anti-activin A antibody or its antigen-binding fragment. In some cases, the antibody or its antigen-binding fragment has a binding-dissociation equilibrium constant (K) less than approximately 5 pM when measured by surface plasmon resonance assay at 25°C. D ) specifically binds to activin A. In some cases, the antibody or its antigen-binding fragment has a K content of less than approximately 4 pM when measured by surface plasmon resonance assay at 25°C. D It specifically binds to activin A. In some cases, the antibody or its antigen-binding fragment has a binding and association equilibrium constant (K) of less than approximately 500 nM. α It specifically binds to activin A.

[0009] In some embodiments, the antibody or its antigen-binding fragment blocks the binding of activin A to at least one activin A receptor. In some embodiments, the antibody or its antigen-binding fragment blocks the activation of at least one activin A receptor by activin A. In some cases, the antibody or its antigen-binding fragment does not significantly block the binding of activin A to activin type II receptors. In some cases, the antibody or its antigen-binding fragment has an IC50 less than approximately 80 pM when measured in an in vivo receptor / ligand-binding bioassay at 25°C. 50The IC50 value blocks the binding of activin A to the activin A receptor. In some cases, the antibody or its antigen-binding fragment has an IC50 of less than approximately 60 pM when measured in an in vivo receptor / ligand-binding bioassay at 25°C. 50 This value blocks the binding of activin A to the activin A receptor.

[0010] In some embodiments, an antibody or its antigen-binding fragment inhibits the binding of activin A, selected from the group consisting of activin type IIA receptor (ActRIIA), activin type IB receptor (ActRIIB), and activin type I receptor, to the activin A receptor. In some embodiments, an antibody or its antigen-binding fragment inhibits activin A-mediated activation of SMAD complex signaling.

[0011] In any of the various embodiments, the antibody or antigen-binding fragment includes (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202, and (b) a CDR of a light chain variable region (LCVR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210. In some embodiments, the antibody or antigen-binding fragment comprises heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210.In some embodiments, the antibody or antigen-binding fragment contains an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, each containing an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 84-86-88-92-94-96, 100-102-104-92-94-96, 108-110-112-92-94-96, 116-1 18-120-92-94-96, 124-126-128-92-94-96, 132-134-136-92-94-96, 140-142-144-148-150-152, 156-158-160-148-150-152, 164-166-168-148-150 -152, 172-174-176-148-150-152, 180-182-184-148-150-152, 188-190-192-148-150-152, 196-198-200-148-150-152 and 204-206-208-212-214-216.

[0012] In any of the various embodiments, the antibody or antigen-binding fragment comprises (a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202, and (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210. In some embodiments, the antibody or antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210.

[0013] In one embodiment, the present invention provides a method for preventing or treating cardiac dysfunction or heart failure in a person in need thereof, comprising administering an antibody or antigen-binding fragment thereof that specifically binds to activin A, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain containing the amino acid sequences of SEQ ID NOs. 68, 70, 72, 76, 78, and 80, respectively. In some embodiments, the antibody or antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NOs. 66 and an LCVR containing the amino acid sequence of SEQ ID NOs. 74.

[0014] In one embodiment, the present invention provides a method for preventing or treating cardiac dysfunction or heart failure in a person in need thereof, comprising administering an antibody or antigen-binding fragment thereof that specifically binds to activin A, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, each comprising the amino acid sequences of SEQ ID NOs: 164-166-168-148-150-152, respectively. In some embodiments, the antibody or antigen-binding fragment comprises HCVR comprising the amino acid sequence of SEQ ID NO: 162 and LCVR comprising the amino acid sequence of SEQ ID NO: 146.

[0015] In any of the various embodiments, the antibody or antigen-binding fragment may be a human antibody containing an IgG heavy chain constant region. In some embodiments, the heavy chain constant region is of the IgG1 isotype. In some embodiments, the heavy chain constant region is of the IgG4 isotype.

[0016] In any of the various embodiments, the method further comprises administering an antibody or antigen-binding fragment in combination with a GDF8 antagonist. In some embodiments, the GDF8 antagonist is selected from the group consisting of a GDF8 inhibitory fusion protein, an anti-GDF8 antibody, and an antigen-binding fragment of an anti-GDF8 antibody. In some cases, the GDF8 antagonist is an anti-GDF8 antibody or its antigen-binding fragment. In some embodiments, the anti-GDF8 antibody comprises a CDR of HCVR containing the amino acid sequence of SEQ ID NO: 217 and a CDR of LCVR containing the amino acid sequence of SEQ ID NO: 221. In some cases, the anti-GDF8 antibody or its antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, each containing the amino acid sequences of SEQ ID NOs: 218-219-220-222-223-224, respectively. In some embodiments, the anti-GDF8 antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 217 and an LCVR having the amino acid sequence of SEQ ID NO: 221.

[0017] In any of the various embodiments described above or herein, the subject is diagnosed with a viral infection. In some embodiments, the viral infection is an infection with a coronavirus. In some cases, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some cases, the subject has severe COVID-19 symptoms. In some cases, the subject has critical COVID-19 symptoms.

[0018] In another embodiment, the present invention provides a pharmaceutical composition comprising an activin A-specific antagonist (e.g., a recombinant human anti-activin A antibody or its antigen-binding fragment as considered above or herein) and a pharmaceutically acceptable carrier for the prevention or treatment of cardiac dysfunction or heart failure in subjects in need thereof.

[0019] In another aspect, the present invention provides an activin A-specific antagonist (e.g., an anti-activin A antibody or its antigen-binding fragment, as discussed above or herein) for use in a method of preventing or treating cardiac dysfunction or heart failure in a person in need thereof.

[0020] In another embodiment, the present invention provides a method for treating COVID-19 in a subject who has tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the method comprising administering an activin A-specific antagonist to the subject. In some embodiments, the activin A-specific antagonist is an anti-activin A antibody or an antigen-binding fragment thereof.

[0021] In some cases, the antibody or antigen-binding fragment contains heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. In some cases, the antibody or its antigen-binding fragment contains an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, each containing an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 84-86-88-92-94-96, 100-102-104-92-94-96, 108-110-112-92-94-96, 116-11 8-120-92-94-96, 124-126-128-92-94-96, 132-134-136-92-94-96, 140-142-144-148-150-152, 156-158-160-148-150-152, 164-166-168-148-150- 152, 172-174-176-148-150-152, 180-182-184-148-150-152, 188-190-192-148-150-152, 196-198-200-148-150-152, and 204-206-208-212-214-216. In some cases, the antibody or antigen-binding fragment comprises (a) an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202, and (b) an LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210.In some cases, the antibody or antigen-binding fragment contains an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. In some cases, the antibody or its antigen-binding fragment contains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, each containing the amino acid sequence of SEQ ID NOs: 68-70-72-76-78-80, respectively. In some cases, the antibody or antigen-binding fragment includes an HCVR containing the amino acid sequence of SEQ ID NO: 66 and an LCVR containing the amino acid sequence of SEQ ID NO: 74. In some cases, the antibody or its antigen-binding fragment includes HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains containing the amino acid sequences of SEQ ID NOs: 164-166-168-148-150-152, respectively. In some cases, the antibody or antigen-binding fragment includes an HCVR containing the amino acid sequence of SEQ ID NO: 162 and an LCVR containing the amino acid sequence of SEQ ID NO: 146.

[0022] In some embodiments, the antibody or antigen-binding fragment is a human antibody containing an IgG heavy chain constant region. In some cases, the heavy chain constant region is of the IgG1 isotype. In some cases, the heavy chain constant region is of the IgG4 isotype.

[0023] In some embodiments, the subjects have severe COVID-19 symptoms requiring oxygen supplementation. In some embodiments, the subjects have very severe COVID-19 symptoms requiring mechanical ventilation or treatment in an intensive care unit.

[0024] In another aspect, the present invention provides the use of an activin A-specific antagonist (e.g., an anti-activin A antibody or its antigen-binding fragment, as discussed above or herein) in the manufacture of a pharmaceutical product for the prevention or treatment of cardiac dysfunction or heart failure in a person in need thereof, or for the treatment of a patient with COVID-19.

[0025] In various embodiments, any combination of features or components of the embodiments discussed above or herein may be used, and such combinations are included within the scope of this disclosure. Any particular value discussed above or herein may be combined with other relevant values ​​discussed above or herein to enumerate ranges in which those values ​​represent upper and lower limits, and such ranges are included within the scope of this disclosure.

[0026] Other embodiments will become apparent from a detailed examination of the embodiments for carrying out the invention, which will be described later. [Brief explanation of the drawing]

[0027] [Figure 1A] This graph shows the negative effect of activin A on the impedance amplitude of human induced pluripotent stem cells in culture medium after a single treatment with activin A (Figure 1A). [Figure 1B] This graph shows the negative effect of activin A on the impedance amplitude of human induced pluripotent stem cells in culture medium after multiple treatments with activin A (Figure 1B). [Figure 2] This graph shows the positive effect of anti-activin A antibody (mAb1) on the prevention of activin A-mediated cardiac dysfunction in human induced pluripotent stem cells. [Figure 3] This is a series of graphs showing the relative increases in activin A, follistatin-related gene (FLRG), and plasminogen activator inhibitor-1 (PAI-1) in serum samples from COVID-19 patients compared to a control. [Figure 4] This is a series of graphs showing the correlation between disease severity and serum levels of activin A and FLRG in COVID-19 patients. [Figure 5] This is a series of graphs showing the relative levels of activin A and FLRG in serum samples from COVID-19 patients of various ages, compared to healthy age-matched controls. [Figure 6] This graph shows the correlation between disease severity and serum PAI-1 levels in COVID-19 patients. [Figure 7] This is a series of graphs showing the correlation between disease severity and serum PAI-1 levels in male (left panel) and female (right panel) COVID-19 patients. [Figure 8] This graph shows the relative levels of PAI-1 in serum samples from COVID-19 patients of various ages, compared to healthy, age-matched controls. [Figure 9] This series of graphs shows that corticosteroid treatment did not significantly affect serum levels of activin A and FLRG in patients with severe or very severe COVID-19 symptoms. [Figure 10] This is a series of graphs showing the activation of cardiac stress gene markers (NPPA - atrial natriuretic peptide, and NPPB - type B natriuretic peptide) and activin A signaling genes (FSTL3 - follistatin-like protein 3, also known as FLRG, and serpin 1, also known as PAI-1) in IPSC-cardiomyocardium treated with activin A. [Figure 11] This graph shows that the IKK / NFKB pathway is primarily involved in activin A induction by IL1β and TNFα. [Figure 12]These are Western blots and graphs showing increased SMAD2 / 3 phosphorylation in human induced pluripotent stem cell (IPSC) cardiomyocytes exposed to activin A, and the blockade of this increased SMAD2 / 3 phosphorylation by an inhibitory anti-activin A antibody (mAb2). The control mAb is an antibody that binds to an unrelated non-human antigen. [Figure 13A] This graph shows the prolongation of action potentials, decrease in electric field potential amplitude, and decrease in electric field potential decline rate in cardiomyocytes chronically exposed to activin A, as well as the prevention of these effects in the presence of an inhibitory anti-activin A antibody (mAb1). [Figure 13B] This graph shows the prolongation of action potentials, decrease in electric field potential amplitude, and decrease in electric field potential decline rate in cardiomyocytes chronically exposed to activin A, as well as the prevention of these effects in the presence of an inhibitory anti-activin A antibody (mAb1). [Figure 14A] This graph shows a decrease in calcium flow peak amplitude, an increase in calcium flow decline time, and an increase in calcium flow rise time in cardiomyocytes chronically exposed to activin A, as well as the prevention of these effects in the presence of an inhibitory anti-activin A antibody (mAb1). [Figure 14B] This graph shows a decrease in calcium flow peak amplitude, an increase in calcium flow decline time, and an increase in calcium flow rise time in cardiomyocytes chronically exposed to activin A, as well as the prevention of these effects in the presence of an inhibitory anti-activin A antibody (mAb1). [Modes for carrying out the invention]

[0028] Before describing the present invention, please understand that the present invention is not limited to such methods and conditions, as the specific methods and experimental conditions described may vary. Also, please understand that the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention, as it is limited only by the appended claims.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains. Where used herein, the term “about” means that, when used in reference to a particular enumerated number, that value may vary by no more than 1% from the enumerated value. For example, where used herein, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0030] Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials will be described below. All patents, applications and non-patent publications referenced herein are incorporated herein by reference in their entirety.

[0031] Methods for preventing and treating cardiac dysfunction and heart failure The present invention provides methods for preventing and treating cardiac dysfunction and heart failure. In some embodiments, the present invention provides methods for treating, preventing, and reducing the severity or progression of heart failure or one or more complications of heart failure. In some aspects, the present invention provides methods for improving human cardiomyocyte function, including contractility and electrical properties.

[0032] As discussed herein, activin A-specific antagonists (e.g., anti-activin A antibodies or their antigen-binding fragments) have been found to offer remarkable effects in the treatment and prevention of various edits of cardiac dysfunction and heart failure. For example, anti-activin A antibodies can be used in a transverse aortic constriction (TAC) heart failure model to prevent or reduce the severity of cardiac hypertrophy, cardiac remodeling, and cardiac fibrosis, as well as to improve cardiac function. In addition, treatment with activin A-specific antagonists can increase the survival time of patients with heart failure. Accordingly, the Disclosure provides, in part, a method of using an activin A-specific antagonist (e.g., an anti-activin A antibody or its antigen-binding fragment) alone or in combination with one or more additional supportive therapies and / or additional active agents to treat, prevent or reduce the severity of heart failure, particularly to treat, prevent or reduce the severity of one or more complications of heart failure (e.g., cardiac hypertrophy, cardiac remodeling, and cardiac fibrosis), and to improve cardiac function and increase the survival time of patients with heart failure.

[0033] As used herein, a therapeutic agent that “prevents” a disorder or symptom means a compound that, in a statistical sample, reduces the occurrence of the disorder or symptom in a treated sample compared to an untreated control sample, or delays the onset of the disorder or symptom compared to an untreated control sample. As used herein, the term “treats” includes, once established, the restoration or elimination of a condition. In either case, prevention or treatment may be identified in a diagnosis made by a physician or other healthcare provider and in the intended outcome of the administration of the therapeutic agent.

[0034] Generally, the treatment or prevention of the diseases or conditions described herein is achieved by administering one or more activin A-specific antagonists (e.g., anti-activin A antibodies or their antigen-binding fragments) in an effective dose. The effective dose of a drug refers to the amount effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome. The therapeutic effective dose of a drug in this disclosure may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the drug's ability to induce the desired response in the individual. The preventive effective dose refers to the amount effective in the dosage and duration required to achieve the desired preventive outcome.

[0035] Heart failure is a clinical syndrome defined by typical symptoms and signs resulting from specific structural or functional abnormalities of the heart (ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. McMurray JJ et al. European Heart Journal 2012, 14(8):803-69, 2013 ACCF / AHA Guideline for the Management of Heart Failure. Yanzy CW et al. Circulation 2013, 128, e240-e327). For example, cardiac abnormalities can impair the ability to fill or empty blood, and / or lead to the inability to deliver enough oxygen to meet the demands of metabolic tissues, despite normal filling pressure, or only at the expense of increased filling pressure. As used herein, the term “heart failure” encompasses a wide range of cardiovascular conditions, including but not limited to heart failure due to left ventricular dysfunction, heart failure with normal ejection fraction, heart failure due to aortic stenosis, acute heart failure, chronic heart failure, congestive heart failure, congenital heart failure, compensated heart failure, decompensated heart failure, diastolic heart failure, systolic heart failure, right-sided heart failure, left-sided heart failure, biventricular heart failure, anterior heart failure, posterior heart failure, high-output heart failure, and low-output heart failure. Heart failure also includes cardiac conditions related to fluid accumulation within the heart, such as myocardial edema.

[0036] Generally, clinical symptoms of heart failure include, for example, dyspnea (shortness of breath), orthopnea, paroxysmal nocturnal dyspnea, fatigue (which may limit exercise), fluid retention (for example, which may lead to pulmonary congestion and peripheral edema), anguina, hypertension, arrhythmias, ventricular arrhythmias, myocardial damage, cardiac hypertrophy, cardiac asthma, nocturia, asthma, congestive liver damage, coagulation disorders, decreased renal blood flow, renal failure, myocardial infarction, and stroke.

[0037] The term "congestive heart failure" is often used to describe all types of heart failure, including those listed above, but it more accurately describes the symptoms of heart failure associated with pulmonary congestion or fluid accumulation in the lungs. This congestion is generally a symptom of systolic and left-sided heart failure. As the efficiency of the pulmonary system decreases, the increased blood volume near the input side of the heart alters the pressure at the alveolar artery interface, i.e., the interface between the pulmonary capillaries and the alveolar spaces of the lungs. This change in pressure at the interface pushes plasma into the alveolar spaces of the lungs. Dyspnea and general fatigue are typical perceived signs of congestive heart failure.

[0038] There are many different ways to classify heart failure. For example, heart failure can be characterized based on which side of the heart is involved (left heart failure vs. right heart failure). Right heart failure impairs pulmonary blood flow to the lungs. Left heart failure impairs aortic blood flow to the body and brain. Mixed presentations are common, and left heart failure often leads to right heart failure over a longer period. Heart failure can also be due to whether the abnormality is due to insufficient contraction (systolic dysfunction, systolic heart failure), or insufficient relaxation of the heart (diastolic dysfunction, diastolic heart failure), or both. Furthermore, heart failure can be classified based on whether the problem is primarily due to increased venous back pressure (preload) or the inability to supply adequate arterial perfusion (afterload). Heart failure can also be classified based on whether the abnormality is due to low cardiac output with high systemic vascular resistance, or high cardiac output with low vascular resistance (low cardiac output heart failure vs. high cardiac output heart failure). Furthermore, heart failure can be classified based on the severity of co-existing conditions, such as heart failure / systemic hypertension, heart failure / pulmonary hypertension, heart failure / diabetes, and heart failure / renal failure.

[0039] Furthermore, heart failure can be classified based on the degree of functional impairment caused by the abnormality of the heart. Functional classification generally relies on the New York Heart Association (NYHA) functional classification. Classes (I-IV) are as follows: Class I: No limitations in any activity, no symptoms in normal activity; Class II: Slight, moderate limitations in activity, patient is comfortable at rest or with light exercise; Class III: Somewhat significant limitations in activity, patient is comfortable only at rest; Class IV: Any physical activity causes discomfort, symptoms occur even at rest. This score can be used to record the severity of symptoms and to assess the response to treatment.

[0040] In the 2001 guidelines, the American College of Cardiology / American Heart Association (ACC) Working Group presented four stages of heart failure (see, for example, Hunt, S., "ACC / AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: A Report of the American College of Cardiology / American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure)," J. Am, Coll. Cardiol, 46:e1-e82:2005). Stage A, the first stage, is for individuals at high risk of heart failure but without structural heart disease or symptoms of heart failure (for example, they are patients with hypertension, atherosclerosis, diabetes, obesity, metabolic syndromes, or those using cardiotoxics). The second stage, Stage B, is for patients with structural heart disease but without signs or symptoms of heart failure (for example, they are patients with a history of myocardial infarction and cardiac remodeling including hypertrophy and low ejection fraction, as well as patients with asymptomatic valvular heart disease). The third stage, Stage C, is for patients with structural heart disease accompanied by past or present symptoms of heart failure (for example, they are patients with known structural heart disease who exhibit shortness of breath and fatigue and have reduced exercise tolerance). The fourth and final stage, Stage D, is for refractory heart failure requiring special intervention (for example, patients with significant symptoms at rest despite maximum drug therapy, i.e., patients who are hospitalized due to relapse or cannot be safely discharged from the hospital without special intervention).The ACC staging system is useful in that Stage A includes "pre-heart failure," i.e., a stage where therapeutic intervention can likely prevent progression to obvious symptoms. ACC Stage A does not have a corresponding NYHA class. ACC Stage B corresponds to NYHA Class I. ACC Stage C corresponds to NYHA Classes II and III, while ACC Stage D overlaps with NYHA Class IV.

[0041] Cardiac remodeling, which typically precedes the clinical signs of heart failure, generally refers to molecular, cellular, and / or interstitial changes that clinically manifest as changes in the size, shape, and function of the heart resulting from cardiac load or injury (Cohn JN et al. JACC 2000. 35(3):569-82). Triggers for cardiac remodeling include, for example, myocardial infarction, hypertension, wall stress, inflammation, pressure overload, and volume overload. Changes in myocardial structure can occur rapidly within hours of injury and can progress over months and years. These changes may initially be beneficial, but over time (from months to years) can impair myocardial function, leading to chronic, refractory heart failure. Notable features of cardiac remodeling include, for example, ventricular dilation, increased ventricular globulus, and the development of interstitial fibrosis and perivascular fibrosis. Increased globulus clearly correlates with mitral regurgitation. Ventricular dilation results primarily from hypertrophy and elongation of cardiomyocytes, and to a lesser extent, from an increase in ventricular mass.

[0042] In some embodiments, activin A-specific antagonists of the present disclosure (e.g., anti-activin A antibodies or their antigen-binding fragments) can be used to treat, prevent, or reduce the progression of cardiac remodeling. For example, activin A-specific antagonists may be used to maintain or reduce changes in the cardiac myocardial structure of a subject. The progression of cardiac remodeling can be evaluated by comparing changes in cardiac myocardial structure over a period of time between two control groups: a first group (treatment group) treated by the method of the present invention, and a second group (placebo group) treated by using a placebo in replacement of or in lieu of the method of the present invention. If the changes in cardiac myocardial structure in the treatment group subjects are smaller than the changes in cardiac myocardial structure in the placebo group subjects, it is determined that there has been a reduction in the progression of cardiac remodeling. Methods for assessing the progression or development of diseases such as cardiac remodeling can be evaluated using well-known methods including, for example, physical examinations, two-dimensional echocardiography combined with Doppler flow imaging, ultrasound, MRI, computed tomography, cardiac catheterization, radionuclide imaging (such as radionuclide ventriculography), and any combination thereof.

[0043] Generally, cardiac remodeling and heart failure arise from disorders and conditions that cause a sustained increase in the load or damage to the heart. Disorders and conditions that can lead to heart failure include, for example, loss of surviving myocardium after myocardial infarction, coronary artery disease, hypertension, cardiomyopathy (e.g., dilated cardiomyopathy, cardiomyopathy due to infection or alcohol / drug abuse, etc.), cardiac valve diseases and dysfunction such as aortic valve disease (e.g., aortic regurgitation, aortic regurgitation, and aortic stenosis), lung disorders (e.g., pulmonary hypertension), congenital heart defects, acute ischemic injury, reperfusion injury, pericardial diseases and abnormalities, myocardial diseases, macrovascular disorders, endocardial disorders, atrial fibrillation, impaired left ventricular myocardial function, impaired right ventricular myocardial function, cardiac arrhythmias, thyroid disorders, renal diseases, diabetes, myocardial weakness that leaves the heart unable to pump enough blood, neurohormonal imbalances, viral infections, and anemia. Because such disorders and symptoms can lead to cardiac remodeling and / or heart failure, subjects having or suspected of having one or more of these conditions are preferred subjects for treatment with one or more activin A-specific antagonists (e.g., anti-activin A antibodies or their antigen-binding fragments) in optional combination with one or more additional active agents or supportive therapies for treating cardiac remodeling and / or heart failure, according to the present invention. In some embodiments, subjects having signs of cardiac remodeling (e.g., myocardial hypertrophy and ventricular dilation) or overt heart failure are also suitable for treatment according to the disclosure, because prevention of further cardiac remodeling, treatment of existing cardiac remodeling, or reduction of cardiac remodeling would be beneficial in these subjects, even if the underlying etiology cannot be detected. In some embodiments, subjects having risk factors for cardiac remodeling and / or heart failure (e.g., subjects having symptoms that can lead to cardiac remodeling and / or chronic heart failure as described herein) are also suitable for treatment according to the disclosure.

[0044] Generally, hypertension or high blood pressure refers to a resting blood pressure exceeding 120 mmHg (systolic) / 80 mmHg (diastolic), for example, when measured with a blood pressure monitor. Blood pressure between 121-139 / 81-89 is considered prehypertension, and anything above this level (140 / 90 mmHg or higher) is considered high (hypertension). Unless otherwise indicated, both prehypertension and hypertension blood pressures are included in the meaning of “hypertension” as used herein. For example, a resting blood pressure of 135 mmHg / 87 or 140 mmHg / 90 mmHg is intended to fall within the range of the term “hypertension,” even though 135 / 87 is generally considered to be in the prehypertension category. Blood pressures of 145 mmHg / 90 mmHg, 140 mmHg / 95 mmHg, and 142 mmHg / 93 mmHg are further examples of hypertension. It will be understood that blood pressure usually fluctuates throughout the day. It can even fluctuate slightly with each heartbeat. It typically rises during activity and falls at rest. It is often higher in cold weather and can rise under stress. More accurate blood pressure readings can be obtained by monitoring blood pressure daily, with readings taken at the same time each day to minimize the influence of external factors. Several readings over time may be needed to determine whether blood pressure is high or not. Generally, chronic hypertension refers to individuals exhibiting high blood pressure continuously or intermittently over long periods, such as at least one week, at least two weeks, at least three weeks, at least four weeks, at least two months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, or at least ten years.

[0045] Generally, cardiac arrhythmia refers to a condition in which the contractions of the heart muscle become irregular. An abnormally fast rhythm (for example, more than 100 beats per minute) is called tachycardia. An abnormally slow rhythm (for example, less than 60 beats per minute) is called bradycardia.

[0046] Generally, cardiac hypertrophy refers to a condition characterized by an increase in the size of the heart and its individual myocardial cells, particularly ventricular myocytes, as well as an increase in the size of the ventricular lumen.

[0047] Ejection fraction is the percentage of blood pumped out of the left ventricle with each heartbeat. It can be measured, for example, during an echocardiogram. Ejection fraction is an important measure of how well the heart is pumping and can be used to classify heart failure and guide treatment. Heart failure can be classified as heart failure with maintained ejection fraction (also called diastolic heart failure) or heart failure with reduced ejection fraction (also called systolic heart failure). Recent studies have demonstrated that the prevalence of heart failure with maintained ejection fraction has increased over a 15-year period without a significant improvement in mortality. If these trends continue, heart failure with maintained ejection fraction may become the most common form of heart failure and is showing signs of becoming a common health problem (Owan et al., 2006, N Engl J Med; 355(3): 251-9).

[0048] In some embodiments, the activin A-specific antagonists of this disclosure may be used to reduce the incidence of non-fatal or fatal cardiovascular events (e.g., myocardial infarction, stroke, angina pectoris, arrhythmia, fluid retention, and progression of heart failure). As used herein, reducing the incidence of cardiovascular events means maintaining or reducing the number of cardiovascular events experienced by a subject over a period of time or over a period of time. The reduction in the incidence of cardiovascular events can be evaluated or determined by comparing the incidence of cardiovascular events over a period of time or between two groups of subjects, where a first group (treatment group) is treated by the method of the present invention and a second group (placebo group) is treated by using a placebo (i.e., a dummy pill) instead of treatment by the method of the present invention. If the number of cardiovascular events in the treatment group is less than the number of cardiovascular events in the placebo group, it is determined that there has been or is not a reduction in the incidence of cardiovascular events. Alternatively, a reduction in the incidence of cardiovascular events can be evaluated or determined by determining the baseline number of cardiovascular events for the target population in a first period, and then measuring the number of cardiovascular events for the target population in a second subsequent period. If the number of cardiovascular events for the target population in the second subsequent period is the same as or less than the number of cardiovascular events for the target population in the first period, it is determined that there has been a reduction in the incidence of cardiovascular events for that target population.

[0049] In some embodiments, the activin A-specific antagonists of this disclosure (e.g., anti-activin A antibodies or their antigen-binding fragments) may be used to reduce the incidence of hospitalization due to heart failure. As used herein, reducing the incidence of hospitalization due to heart failure means maintaining or decreasing the number of hospitalizations due to heart failure experienced by a subject over a period of time or over a period of time. The reduction in the incidence of hospitalization due to heart failure can be evaluated or determined by comparing the incidence of hospitalization due to heart failure over a period of time or during a period of time between two groups of subjects, one group (treatment group) treated by the method of the present invention and the other group (placebo group) treated by using a placebo (i.e., a dummy pill) instead of treatment by the method of the present invention. If the number of hospitalizations due to heart failure in the treatment group is less than the number of hospitalizations due to heart failure in the placebo group, it is determined that there has been or is not a reduction in the incidence of hospitalization due to heart failure. Alternatively, a decrease in the incidence of hospitalization due to heart failure can be evaluated or determined by determining the baseline number of hospitalizations due to heart failure in the target population during the first period, and then measuring the number of hospitalizations due to heart failure in the target population during the second subsequent period. If the number of hospitalizations due to heart failure in the target population during the second subsequent period is less than or equal to the number of hospitalizations due to heart failure in the target population during the first period, it is determined that the incidence of hospitalization due to heart failure in the target population has decreased.

[0050] In some embodiments, the activin A-specific antagonists of this disclosure (e.g., anti-activin A antibodies or their antigen-binding fragments) may be used to improve the survival of patients with heart failure. As used herein, improving the survival of patients with heart failure means maintaining or reducing the number of fatal cardiovascular events experienced by a target population over a period of time or over a period of time. Improvement in the survival of patients with heart failure can be evaluated or determined by comparing the incidence of fatal cardiovascular events over a period of time or during a period of time between two target groups, where a first group (treatment group) is treated by the method of the present invention and a second group (placebo group) is treated by using a placebo (i.e., a dummy pill) instead of treatment by the method of the present invention. If the number of fatal cardiovascular events in the treatment group is less than the number of fatal cardiovascular events in the placebo group, it is determined that there has been or is not an improvement in the survival of patients with heart failure. Alternatively, a reduction in the incidence of fatal cardiovascular events can be evaluated or determined by determining the baseline number of fatal cardiovascular events for the target population in a first period, and then measuring the number of fatal cardiovascular events for the target population in a second subsequent period. If the number of fatal cardiovascular events for the target population in the second subsequent period is the same as or less than the number of fatal cardiovascular events for the target population in the first period, it is determined that there has been an improvement in the survival rate of heart failure patients in that target population.

[0051] In some embodiments, the activin A-specific antagonists of this disclosure (e.g., anti-activin A antibodies or their antigen-binding fragments) may be used to reduce the risk of cardiovascular death in patients with heart failure. As used herein, reducing the risk of cardiovascular death in patients with heart failure means maintaining or reducing the number of fatal cardiovascular events experienced by a target population over a period of time or over a period of time. A reduction in cardiovascular death in patients with heart failure can be evaluated or determined by comparing the incidence of fatal cardiovascular events over a period of time or during a period of time between two groups of subjects, one group (treatment group) treated by the method of the present invention and the other group (placebo group) treated by using a placebo (i.e., a dummy pill) instead of treatment by the method of the present invention. If the number of fatal cardiovascular events in the treatment group is less than the number of fatal cardiovascular events in the placebo group, it is determined that there has been or is not a reduction in cardiovascular death in patients with heart failure. Alternatively, a reduction in cardiovascular death in heart failure patients can be evaluated or determined by determining the baseline number of fatal cardiovascular events in the target population during a first period, and then measuring the number of fatal cardiovascular events in the target population during a second, subsequent period. If the number of fatal cardiovascular events in the target population during the second, subsequent period is equal to or less than the number of fatal cardiovascular events in the target population during the first period, it is determined that there has been a reduction in cardiovascular death in heart failure patients in that target population.

[0052] In some embodiments, the activin A-specific antagonists of this disclosure (e.g., anti-activin A antibodies or their antigen-binding fragments) may be used to treat cardiac dysfunction in patients with confirmed SARS-CoV-2 virus infection and one or more symptoms of COVID-19, such as fever, cough, or shortness of breath. Patients with COVID-19 and pre-existing cardiovascular disease have been reported to be at high risk of serious illness and death. Furthermore, SARS-CoV-2 infection has been associated with several direct and indirect cardiovascular complications, including acute myocardial injury, myocarditis, arrhythmias, and venous thromboembolism (Driggin et al., J Am Coll Cardiol., 75(18):2352-2371, May 2020). A hyperinflammatory response associated with the production of large amounts of pro-inflammatory cytokines and chemokines has also been reported in COVID-19 patients (Soy et al., Clinical Rheumatology, doi.org / 10.1007 / s10067-020-05190-5, May 2020). While not intended to be theoretically bound, increased cytokine production may activate increased activin A expression, which in turn reduces cardiomyocyte contractile amplitude, slows contractile dynamics, impairs calcium processing in cardiomyocytes, leading to cardiac dysfunction and, in some cases, heart failure.

[0053] A wide variety of approved medications and supportive therapies are currently used to manage patients with heart failure and those at risk of heart failure (e.g., patients with hypertension, dyslipidemia, diabetes, and vascular disorders). Such medications include, for example, adrenergic blockers (alpha-blockers and beta-blockers), centrally acting alpha-agonists, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, positive inotropes, vasodilators, benzodiazepines, renin inhibitors, antithrombotic agents, and various types of diuretics (e.g., loop, potassium-sparing, thiazide, and thiazide-like). Surgical treatments for treating or preventing heart failure include, for example, physical procedures to increase the internal size of constricted arteries, such as balloon angioplasty or stenting. In some embodiments, the present disclosure provides a method for treating heart failure or one or more complications of heart failure, comprising the step of administering an activin A-specific antagonist (e.g., an anti-activin A antibody or its antigen-binding fragment) in combination with additional active agents or supportive therapies (e.g., adrenergic blockers, centrally acting α-agonists, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, systolic enhancers, diuretics, and various surgical treatments) for treating, preventing, or reducing the progression of heart failure.

[0054] Activin A-specific antagonist The method of the present invention utilizes an activin A-specific antagonist, such as an activin A-specific binding protein, an activin A small molecule inhibitor, or an activin A nucleotide antagonist.

[0055] As used herein, the term “antigen-specific binding protein” means a protein that contains at least one domain that specifically binds to a particular antigen. Exemplary categories of antigen-specific binding proteins include antibodies, antigen-binding portions of antibodies, peptides (e.g., peptide bodies) that specifically interact with a particular antigen, receptor molecules that specifically interact with a particular antigen but not with other antigens, and proteins that contain ligand-binding portions of receptors that specifically bind to a particular antigen but not with other antigens.

[0056] The method of the present invention involves the use of an antigen-specific binding protein that specifically binds to activin A, i.e., an "activin A-specific binding protein." Activin is a homodimer molecule comprising beta subunits (i.e., inhibin βA, inhibin βB, inhibin βC, and / or inhibin βE). The βA subunit comprises the amino acid sequence of SEQ ID NO: 226, and the βB subunit comprises the amino acid sequence of SEQ ID NO: 228. Activin A is a homodimer of two βA subunits. Activin B is a homodimer of two βB subunits. Activin AB is a heterodimer of one βA subunit and one βB subunit, and Activin AC is a heterodimer of one βA subunit and one βC subunit. An activin A-specific binding protein may be an antigen-specific binding protein that specifically binds to the βA subunit. Since the βA subunit is present in activin A, activin AB, and activin AC molecules, an "activin A-specific binding protein" can be an antigen-specific binding protein that specifically binds to activin A, as well as activin AB and activin AC (through its interaction with the βA subunit). Therefore, according to one embodiment of the present invention, an activin A-specific binding protein specifically binds to activin A; or activin A and activin AB; or activin A and activin AC; or activin A, activin AB, and activin AC, but does not bind to other ActRIIB ligands such as activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, and Nodal. Thus, in one embodiment of the present invention, an activin A-specific binding protein specifically binds to activin A, but does not significantly bind to activin B or activin C. In another embodiment, the activin A-specific binding protein may also bind to activin B (by cross-reaction with the βB subunit, i.e., inhibin βB). In yet another embodiment, the activin A-specific binding protein is a binding protein that specifically binds to activin A but does not bind to any other ligand of ActRIIB.In another embodiment, the activin A-specific binding protein is a binding protein that specifically binds to activin A but does not bind to any bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP4, BMP6, BMP9, BMP10). In yet another embodiment, the activin A-specific binding protein is a binding protein that specifically binds to activin A but does not bind to any other member of the transforming growth factor beta (TGFβ) superfamily.

[0057] Some embodiments of the methods of the present invention also include an antigen-specific binding protein that specifically binds to GDF8 (i.e., a “GDF8-specific binding protein”). The term “GDF8” (also known as “growth and differentiation factor-8” and “myostatin”) refers to the protein (mature protein) having the amino acid sequence of SEQ ID NO: 225. According to these embodiments, the GDF8-specific binding protein specifically binds to GDF8 but does not bind to other ActRIIB ligands, such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, activin A, activin B, activin AB, Nodal, etc.

[0058] In the context of the present invention, molecules such as ActRIIB-Fc (e.g., "ACE-031" or "RAP-031"), which contain the ligand-binding portion of the ActRIIB receptor, are not considered "activin A-specific binding proteins" or "GDF8-specific binding proteins" because such molecules bind to multiple ligands in addition to GDF8, activin A, and activin AB.

[0059] All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of each protein, polypeptide, or protein fragment, unless explicitly identified as originating from a non-human species.

[0060] specific binding When used herein, terms such as "specifically bind" refer to an antigen-specific binding protein or antigen-specific binding domain having a dissociation constant (K) of 500 pM or less. D This refers to a characteristic of forming a complex with a specific antigen that does not bind to other unrelated antigens under normal test conditions. An "unrelated antigen" is a protein, peptide, or polypeptide that has less than 95% amino acid identity with each other. Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. For example, the antigen-specific binding protein or antigen-specific binding domain used in connection with the present invention has a K content of less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, less than approximately 100 pM, less than approximately 90 pM, less than approximately 80 pM, less than approximately 70 pM, less than approximately 60 pM, less than approximately 50 pM, less than approximately 40 pM, less than approximately 30 pM, less than approximately 20 pM, less than approximately 10 pM, less than approximately 5 pM, less than approximately 4 pM, less than approximately 2 pM, less than approximately 1 pM, less than approximately 0.5 pM, less than approximately 0.2 pM, less than approximately 0.1 pM, or less than approximately 0.05 pM when measured by surface plasmon resonance assay. D It contains molecules that bind to specific antigens (activin A and / or AB, or GDF8).

[0061] When used herein, an antigen-specific binding protein or antigen-specific binding domain is tested for binding to a molecule at 25°C in a surface plasmon resonance assay, and exhibits a K+50.0 nM binding. D When such assay or equivalent shows no binding, it is said that a particular molecule "does not bind" (e.g., "does not bind to GDF11," "does not bind to BMP9," "does not bind to BMP10," etc.).

[0062] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of interactions, for example, by using a BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ), through detection of changes in protein concentration within a biosensor matrix.

[0063] The term "K D ", as used herein, means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, the K D values disclosed herein refer to K D values determined by surface plasmon resonance assays at 25°C.

[0064] Antibodies and antigen-binding fragments An antigen-specific binding protein can comprise or consist of an antibody or an antigen-binding fragment of an antibody. Further, in the case of an antigen-binding molecule that contains two different antigen-specific binding domains (described below), one or both of the antigen-specific binding domains can comprise or consist of an antigen-binding fragment of an antibody.

[0065] As used herein, "antibodies that bind activin" or "anti-activin A antibodies" include antibodies and antigen-binding fragments thereof that bind to soluble fragments of activin A protein and can also bind to activin heterodimers containing the activin βA subunit.

[0066] As used in the present invention, the term "antibody" means any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (for example, activin A). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy-chain variable region (referred to herein as HCVR or VH It includes the heavy chain constant region (abbreviated as C). The heavy chain constant region consists of three domains, C H 1, C H 2, and C H Includes 3. Each light chain has a light chain variable region (LCVR or V in this specification). L It includes a light chain steady region (abbreviated as C). The light chain steady region consists of one domain (C L Includes 1). V H Region and V L The region can be further subdivided into a highly variable region called the Complementarity Determination Region (CDR), which contains more conserved regions called the framework region (FR). H and V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-activin A antibody (or its antigen-binding moiety) may be identical to the human germline sequence, or they may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0067] As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. “Antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and similar terms, as used herein, include natural, enzymatically available, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Antigen-binding fragments of antibodies may originate from complete antibody molecules using any suitable standard technique, such as protein digestion techniques or recombinant genetic engineering techniques, which may involve the manipulation and expression of the DNA-coding antibody variable domain and optionally the constant domain. Such DNA is known and / or readily available, for example, from commercially available sources, DNA libraries (e.g., including phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by molecular biological techniques to, for example, position one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0068] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or restricted FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included within the expression "antigen-binding fragment" as used herein.

[0069] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. H Domain is V L In antigen-binding fragments associated with the domain, V H Domain and V L The domains may be positioned relative to each other in any preferred arrangement. For example, the variable region is a dimer, V H -V H , V H -V L , or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may be a monomer V H Domain or V L You may include a domain name.

[0070] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention include (i)V H -C H 1. (ii)V H -C H 2, (iii)V H -C H 3, (iv)V H -C H 1-C H 2. (v)V H -C H 1-C H 2-C H 3. (vi)V H -C H 2-C H 3. (vii)V H -C L (viii)V L -C H 1. (ix)V L -C H 2, (x)V L -C H 3. (xi)VL -C H 1-C H 2. (xii)V L -C H 1-C H 2-C H 3. (xiii)V L -C H 2-C H 3, and (xiv)V L -C L These include any of the exemplary configurations listed above for the variable domain and the constant domain, in which the variable domain and the constant domain may be directly linked to each other or linked by a complete or partial hinge region or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in flexible or semi-flexible linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may be linked to each other and / or one or more monomers V H Or V L In non-covalent bonds with the domain (e.g., via disulfide bonds), the domain may contain a homodimer or heterodimer (or other polymer) among the variable domain configurations and constant domain configurations listed above.

[0071] Similar to complete antibody molecules, antibody-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two distinct variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody form, including the exemplary bispecific antibody forms disclosed herein, can be adapted for use in association with the antibody antigen-binding fragments of the present invention using conventional techniques available in the art.

[0072] In certain embodiments of the present invention, the anti-activin A antibody of the present invention is a human antibody. As used in the present invention, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include, for example, amino acid residues in the CDR, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0073] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (as described later), antibodies isolated from recombinant combinatorial human antibody libraries (as described later), antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (e.g., Taylor et al., Nucl Acids Res 20:6287-6295 (1992)), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic to human Ig sequences are used), and therefore the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V LThese sequences are derived from and related to other sequences, but they are sequences that cannot naturally exist in the human antibody germline repertoire in vivo.

[0074] Human antibodies can exist in two forms related to hinge heterogeneity. In the first form, the immunoglobulin molecule contains a stable quadruple-chain construct of approximately 150-160 kDa, where the dimers are held together by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, and a molecule of approximately 75-80 kDa is formed, consisting of covalently bonded light and heavy chains (half-antibodies). These forms are considered extremely difficult to separate, even after affinity purification.

[0075] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using the human IgG1 hinge (Angal et al. Molecular Immunology 30:105 1993). This invention relates to the hinge region, C H 2 regions, or C H This includes antibodies having one or more mutations in three regions, which may be desirable, for example, to improve the yield of a desired antibody morphology during production.

[0076] The antibodies of the present invention may be isolated antibodies. “Isolated antibodies,” as used herein, mean antibodies identified, isolated and / or recovered from at least one component of their natural environment. For example, antibodies isolated or removed from at least one component of an organism, or from tissues or cells in which antibodies are naturally present or naturally produced, are “isolated antibodies” for the purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies subjected to at least one purification or isolation step. According to certain embodiments, isolated antibodies may substantially contain no other cellular material and / or chemical substances.

[0077] The present invention includes the neutralization and / or blocking of anti-activin A antibodies. “Neutralizing” or “blocking” antibodies, as used herein, are intended to refer to those whose binding to activin A is as follows: (i) interfere with the interaction between activin A and the activin A receptor (e.g., activin type IIA receptor, activin type IIB receptor, activin type I receptor, etc.), (ii) inhibit the formation of the activin-activin receptor complex, and / or (iii) consequently inhibit at least one biological function of activin A. The inhibition caused by an activin A neutralizing or blocking antibody does not need to be complete, as long as it is detectable using a suitable assay. Exemplary assays for detecting activin A inhibition are described in the examples herein.

[0078] The anti-activin A antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention comprises antibodies and antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to corresponding residues in the germline sequence from which the antibody is derived, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LIn other embodiments, all of the framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody originated. In other embodiments, only specific residues are mutated back to the original germline sequence, for example, the mutated residues are found within the first eight amino acids of FR1 or within the last eight amino acids of FR4, or the mutated residues are found only within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while other specific residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies and antibody-conjugated fragments containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced (in some cases) biological properties of antagonists or agonists, or decreased immunogenicity. Antibodies and antibody-conjugated fragments obtained by this general method are included within the scope of the present invention.

[0079] The present invention also includes anti-activin A antibodies comprising variants of any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, the present invention includes anti-activin A antibodies comprising HCVR, LCVR, and / or CDR amino acid sequences having conservative amino acid substitutions, such as 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer, associated with any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0080] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl groups on an antigen.

[0081] The terms “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicate that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), it has at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0082] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned using predefined gap weights by programmed GAP or BESTFIT, etc. Preferably, the non-identical residue positions differ in their conserved amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If the conservative substitutions of two or more amino acid sequences are different from each other, the sequence identity percentage or degree of similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson, WR, Methods Mol Biol 24:307-331 (1994), incorporated herein by reference. Examples of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-1445 (1992), which is incorporated herein by reference. A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0083] Sequence similarity to polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the best overlap region alignment and sequence identity percentage between the query sequence and the search sequence (see, for example, Pearson, WR, Methods Mol Biol 132:185-219 (2000), incorporated herein by reference). Another preferred algorithm for comparing the sequences of the present invention with a database containing numerous sequences from different biological origins is the computer program BLAST, particularly BLASTP or TBLASTN, which uses default parameters. See, for example, Altschul et al., J Mol Biol 215:403-410 (1990) and Altschul et al., Nucleic Acids Res 25:3389-402 (1997), which are incorporated herein by reference, respectively.

[0084] The present invention provides an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity substantially similar thereto.

[0085] The present invention further provides an antibody or an antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0086] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCVR and LCVR (HCVR / LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210.

[0087] The present invention also provides an antibody or antigen-binding fragment thereof comprising: a heavy chain CDR3 (HCDR3) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 112, 120, 128, 136, 144, 160, 168, 176, 184, 192, 200, and 208, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 152, and 216, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0088] In a particular embodiment, the antibody or the antigen-binding portion of the antibody comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 96, 112 / 96, 120 / 96, 128 / 96, 136 / 96, 144 / 152, 160 / 152, 168 / 152, 176 / 152, 184 / 152, 192 / 152, 200 / 152, and 208 / 216.

[0089] The present invention also relates to a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 108, 116, 124, 132, 140, 156, 164, 172, 180, 188, 196, and 204, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 110, 118, 126, 134, 142, 158, 166, 174, 182, 190, 198, and 206, or having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity The present invention provides an antibody or fragment thereof further comprising a heavy chain CDR2 (HCDR2) domain having a substantially similar sequence, an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 148, and 212, or a light chain CDR1 (LCDR1) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 150, and 214, or a light chain CDR2 (LCDR2) domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0090] The specific, non-limiting, and exemplary antibody and antigen-binding fragments of the present invention each contain an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g., H4H10423P), 20-22-24-28-30-32 (e.g., H4H10424P), 36-38-40-44-46-48 (e.g., H4H10 426P), 52-54-56-60-62-64 (e.g., H4H10429P), 68-70-72-76-78-80 (e.g., H4H10430P), 84-86-88-92-94-96 (e.g., H4H10432P2), 100-102-104-92-94-96 (e.g., H4H10433P2), 108-110-112-92-94-96 (e.g., H4H10436P2), 116-118-120-92-94 -96 (e.g., H4H10437P2), 124-126-128-92-94-96 (e.g., H4H10438P2), 132-134-136-92-94-96 (e.g., H4H10440P2), 140-142-144-148-150-152 (e.g., H4H10442P2), 156-158-160-148-150-152 (H4H10445P2), 164-166-168-148-150-152 (H4 H10446P2), 172-174-176-148-150-152 (H4H10447P2), 180-182-184-148-150-152 (H4H10448P2), 188-190-192-148-150-152 (H4H10452P2), 196-198-200-148-150-152 (H4H10468P2), and 204-206-208-212-214-216 (H2aM10965N).In some embodiments, the anti-activin A antibody comprises an HCVR and / or LCVR that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the above-mentioned CDR sequence and the corresponding HCVR and LCVR (HCVR / LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210.

[0091] In some embodiments, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to activin A, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within a heavy chain variable region and light chain variable region (HCVR / LCVR) sequence selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. Methods and techniques for identifying CDRs within HCVR amino acid sequences and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within specific HCVR amino acid sequences and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., PNAS (USA) 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0092] The present invention includes anti-activin A antibodies containing modified hydrocarbons. In some applications, modifications to remove undesirable glycosylation sites may be useful. In some applications, antibodies with modifications to remove undesirable glycosylation sites, or antibodies with deletions of fucose moieties present on oligosaccharide chains to enhance antibody-dependent cellular cytotoxicity (ADCC) function, for example, may be useful (see Shield et al. J Biol Chem 277:26733 (2002)). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC). In some applications, antibodies may have modified glycosylation patterns to minimize effector function. For example, antibodies may be modified to obtain further glycosylated or sialylated antibodies.

[0093] Biological characteristics of antibodies The present invention includes anti-activin A antibodies and antigen-binding fragments thereof that bind to activin A with high affinity. For example, the present invention includes antibodies that, when measured by surface plasmon resonance using an assay format as defined in the examples herein, have a K content of less than approximately 30 nM. DThe present invention includes antibodies and antigen-binding fragments of antibodies that bind to activin A (for example, at 25°C or 37°C). In certain embodiments, the antibodies or antigen-binding fragments of the present invention, when measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or substantially similar assays, have a molecular weight of less than approximately 25 nM, less than approximately 20 nM, less than approximately 15 nM, less than approximately 10 nM, less than approximately 5 nM, less than approximately 2 nM, less than approximately 1 nM, less than approximately 500 pM, less than approximately 250 pM, less than approximately 240 pM, less than approximately 230 pM, less than approximately 220 pM, less than approximately 210 pM, less than approximately 200 pM, less than approximately 190 pM, less than approximately 180 pM, less than approximately 170 pM, and less than approximately 160 pM. K levels below approximately 150 pM, below approximately 140 pM, below approximately 130 pM, below approximately 120 pM, below approximately 110 pM, below approximately 100 pM, below approximately 95 pM, below approximately 90 pM, below approximately 85 pM, below approximately 80 pM, below approximately 75 pM, below approximately 70 pM, below approximately 65 pM, below approximately 60 pM, below approximately 55 pM, below approximately 50 pM, below approximately 45 pM, below approximately 40 pM, below approximately 35 pM, below approximately 30 pM, below approximately 25 pM, below approximately 20 pM, below approximately 15 pM, below approximately 10 pM, below approximately 9 pM, below approximately 8 pM, below approximately 7 pM, below approximately 6 pM, below approximately 5 pM, below approximately 4 pM, or below approximately 3 pM. D It then binds to activin A.

[0094] The present invention also includes anti-activin A antibodies and their antigen-binding fragments that inhibit activin A-mediated cellular signaling. For example, the present invention includes IC50 less than about 4 nM when measured in a cell-based blocking bioassay using, for example, the assay format defined in the examples herein, or substantially similar assays. 50The present invention includes anti-activin A antibodies that inhibit the activation of the SMAD complex signaling pathway via the binding of activin A to activin type I or II receptors, in terms of value. In certain embodiments, the antibodies or antigen-binding fragments of the present invention, when measured in a cell-based blocking bioassay using, for example, the assay format defined in the examples herein, or substantially similar assays, inhibit the activation of the SMAD complex signaling pathway via the binding of activin A to activin type I or II receptors in the following ranges: less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, less than 250 pM, less than 240 pM, less than 230 pM, less than 220 pM, less than 210 pM, less than 200 pM, less than 190 pM, and less than 18 ICs with a capacitance of less than 0 pM, less than approximately 170 pM, less than approximately 160 pM, less than approximately 150 pM, less than approximately 140 pM, less than approximately 130 pM, less than approximately 120 pM, less than approximately 110 pM, less than approximately 100 pM, less than approximately 95 pM, less than approximately 90 pM, less than approximately 85 pM, less than approximately 80 pM, less than approximately 75 pM, less than approximately 70 pM, less than approximately 65 pM, less than approximately 60 pM, less than approximately 55 pM, less than approximately 50 pM, less than approximately 49 pM, less than approximately 48 pM, less than approximately 47 pM, less than approximately 46 pM, less than approximately 45 pM, less than approximately 44 pM, less than approximately 43 pM, less than approximately 42 pM, less than approximately 41 pM, less than approximately 40 pM, or less than approximately 39 pM. 50 Inhibits by value. In certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits activin B signaling by interfering with the binding of activin B to activin type I or type II receptors, thereby inhibiting IC 50The values ​​are less than approximately 50 nM, less than approximately 20 nM, less than approximately 10 nM, less than approximately 5 nM, or less than approximately 1 nM when measured in a cell-based blocking bioassay using, for example, the assay format defined in the examples herein or substantially similar assays. In certain embodiments, the antibody or antigen-binding fragment of the present invention activates the SMAD complex signaling pathway via the binding of activin AB to activin type I or II receptors, when measured in a cell-based blocking bioassay using, for example, the assay format defined in the examples herein or substantially similar assays, and the values ​​are less than approximately 500 pM, less than approximately 450 pM, less than approximately 440 pM, less than approximately 430 pM, less than approximately 420 pM, less than approximately 410 pM, less than approximately 400 pM, or less than approximately 390 pM. ICs with a pressure of approximately 140 pM or less: 140 pM, less than approximately 380 pM, less than approximately 370 pM, less than approximately 360 pM, less than approximately 350 pM, less than approximately 340 pM, less than approximately 320 pM, less than approximately 310 pM, less than approximately 300 pM, less than approximately 290 pM, less than approximately 280 pM, less than approximately 270 pM, less than approximately 260 pM, less than approximately 250 pM, less than approximately 240 pM, less than approximately 230 pM, less than approximately 220 pM, less than approximately 210 pM, less than approximately 200 pM, less than approximately 190 pM, less than approximately 180 pM, less than approximately 170 pM, less than approximately 160 pM, less than approximately 150 pM, or less than approximately 140 pM. 50 Inhibits by value. In certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits the activation of the SMAD complex signaling pathway via the binding of activin AC to activin type I or II receptors when measured in a cell-based blocking bioassay using, for example, the assay format defined in the examples herein or substantially similar assays, with IC values ​​of less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 750 pM, less than about 700 pM, less than about 650 pM, less than about 600 pM, or less than about 580 pM. 50 Inhibit it with a value.

[0095] The antibodies of the present invention may have one or more of the biological characteristics described above, or any combination thereof. Other biological characteristics of the antibodies of the present invention will become apparent to those skilled in the art from a further examination of this disclosure, including the examples provided herein.

[0096] Anti-activin A antibodies containing Fc variants According to a particular embodiment of the present invention, an anti-activin A antibody is provided which comprises an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides an Fc domain containing C H 2 or C H The anti-activin A antibody contains mutations in three regions, which increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-restrictive examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0097] For example, the present invention includes 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), 428L and 434S (e.g., M428L and N434S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and The anti-activin A antibody includes an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domain disclosed herein are considered within the scope of the present invention.

[0098] The present invention also includes anti-activin A antibodies containing a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH region of one or more immunoglobulin isotypes. For example, the antibody of the present invention may include a chimeric CH region comprising part or all of the CH2 domain derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule, combined with part or all of the CH3 domain derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule. According to a particular embodiment, the antibody of the present invention includes a chimeric CH region having a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues at EU numbering positions 216-227) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at EU numbering positions 228-236) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See U.S. Provisional Patent Application No. 61 / 759,578, filed February 1, 2013, the entire disclosure of which is incorporated herein by reference).

[0099] Epitope mapping and related techniques The present invention includes an anti-activin A antibody that interacts with one or more amino acids present within activin A (e.g., within the activin type II receptor binding site). The epitope to which the antibody binds may consist of a single continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) located within the activin βA subunit. Alternatively, the epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) located within the activin A dimer.

[0100] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, the routine cross-blocking assay described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, Methods Mol Biol 248:443-463 (2004)), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, Protein Science 9:487-496 (2000)). Another method that can be used to identify the amino acids within the polypeptide with which the antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange methods involve deuterizing the protein of interest and then conjugating the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to induce hydrogen-deuterium exchange at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring, Analytical Biochemistry 267(2):252-259 (1999) and Engen and Smith, Anal. Chem. 73:256A-265A (2001).

[0101] The present invention further includes anti-activin A antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, H2aM10965N, etc.). Similarly, the present invention also includes anti-activin A antibodies that compete with any of the specific exemplary antibodies described herein for binding to activin A (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, H2aM10965N, etc.). For example, the present invention includes anti-activin A antibodies that cross-compete with one or more antibodies exemplified herein (e.g., H4H10423P, H4H10446P2, H4H10468P2, and H4H10442P2) for binding to activin A. The present invention also includes anti-activin A antibodies that cross-compete with one or more antibodies such as H4H10429, H4H1430P, H4H10432P2, H4H10436P2, and H4H10440P2 for binding to activin A.

[0102] By using methods readily known in the art and commonly illustrated herein, it is possible to easily determine whether an antibody binds to the same epitope as the reference anti-activin A antibody, or whether it competes with the reference anti-activin A antibody for binding. For example, to determine whether a test antibody binds to the same epitope as the reference anti-activin A antibody of the present invention, the reference antibody is conjugated to activin A (or a βA subunit-containing heterodimer) under saturated conditions. Next, the ability of the test antibody to bind to the activin A molecule is evaluated. If the test antibody can bind to activin A after saturated binding with the reference anti-activin A antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-activin A antibody. On the other hand, if the test antibody cannot bind to the activin A protein after saturated binding with the reference anti-activin A antibody, the test antibody may bind to the same epitope as the epitope conjugated by the reference anti-activin A antibody of the present invention. Next, further conventional experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed deletion of the test antibody binding is actually due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the observed deletion of binding. These types of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, for example, two antibodies bind to the same (or overlapping) epitopes if, when measured in a competitive binding assay, one antibody in 1, 5, 10, 20, or 100-fold excess inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 50:1495-1502 (1990)). Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope.Two antibodies are considered to have a "duplicate epitope" if only a subset of amino acid mutations that reduces or eliminates the binding of one antibody reduces or eliminates the binding of the other antibody.

[0103] To determine whether an antibody competes for (or cross-competes for) binding to a reference anti-activin A antibody, the binding methodology described above is performed in two directions. In the first direction, the reference antibody is bound to the activin A protein (or a dimer containing the βA subunit) under saturated conditions, and then the binding of the test antibody to the activin A molecule is evaluated. In the second direction, the test antibody is bound to activin A under saturated conditions, and then the binding of the reference antibody to activin A is evaluated. If, of both directions, only the first (saturated) antibody can bind to the activin A molecule, it is concluded that the test antibody and the reference antibody compete for binding to activin A. As will be recognized by those skilled in the art, an antibody competing for binding to a reference antibody does not necessarily need to bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0104] The anti-activin A antibody of the present invention can bind to an epitope on activin A within or near its binding site to the activin type II receptor, directly blocking the interaction between activin A and the activin type II receptor, and indirectly blocking the interaction between activin A and the activin type I receptor. The anti-activin A antibody of the present invention can bind to an epitope on activin A within or near its binding site to the activin type I receptor, directly blocking the interaction between activin A and the activin type I receptor. In one embodiment of the present invention, the anti-activin A antibody of the present invention that binds to activin A at or near its activin type I receptor binding site does not block the interaction between activin A and the activin A type II receptor.

[0105] Preparation of human antibodies Methods for producing monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Some such known methods can be used in the context of the present invention to produce human antibodies that specifically bind to human activin A.

[0106] For example, using VELOCIMMUNE™ technology or any other known method for generating fully human monoclonal antibodies, a high-affinity chimeric antibody against human activin A having a human variable region and a mouse constant region is first isolated. The antibody is then characterized and selected for desirable features, including affinity, selectivity, and epitopes, as described in the experimental section below. If necessary, the mouse constant region is replaced with a desired human constant region, e.g., wild-type or modified IgG1 or IgG4, to generate a fully human anti-activin A antibody. The selected constant region may vary depending on the specific application, but the high-affinity antigen-binding features and target-specific features reside in the variable region. In certain examples, the fully human anti-activin A antibody is isolated directly from antigen-positive B cells.

[0107] biological equivalent The anti-activin A antibodies and antibody fragments of the present invention comprise proteins having different amino acid sequences than those of the described antibodies but retaining the ability to bind to activin A. Such mutant antibodies and antibody fragments, compared to the parent sequence, involve the addition, deletion, or substitution of one or more amino acids, but exhibit biological activity essentially equivalent to that of the described antibodies. Similarly, the DNA sequences encoding the anti-activin A antibodies of the present invention comprise sequences encoding anti-activin A antibodies or antibody fragments that, compared to the disclosed sequences, involve the addition, deletion, or substitution of one or more nucleotides, but are essentially biologically equivalent to the anti-activin A antibodies or antibody fragments of the present invention. Examples of such mutant amino acid and DNA sequences are discussed above.

[0108] Two antigen-binding proteins or antibodies are considered bioequivalents or pharmacologic alternatives if, for example, they are administered at the same molar dose, either as a single dose or multiple doses, under similar experimental conditions, and their absorption rates and degrees of absorption do not show significant differences. Some antibodies are considered equivalent or pharmacologic substitutes if they are equivalent in their absorption degrees but not in their absorption rates, and such differences in absorption rates are intentional and reflected in labeling, and therefore may be considered bioequivalent. These are considered not essential for achieving effective bodily drug concentrations for long-term use and are not medically significant for the particular drug product being studied.

[0109] In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference in their safety, purity, or efficacy.

[0110] In one embodiment, the two antigen-binding proteins are bioequivalent if the patient can be switched between them once or more, compared to a therapy sustained without switching between a reference product and a biological product, without an expected increase in the risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy.

[0111] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by a common mechanism or mechanism of action with respect to the conditions or conditions of use, to the extent that such mechanism is known.

[0112] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) reasonably predictive in vitro studies that correlate with human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0113] Biologically equivalent variants of the anti-activin A antibody of the present invention can be constructed, for example, by causing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other contexts, biologically equivalent antibodies may include antibody variants that involve amino acid changes that modify the glycosylation characteristics of the anti-activin A antibody, such as mutations that eliminate or remove glycosylation.

[0114] Species selectivity and species cross-reactivity The present invention provides, according to certain embodiments, anti-activin A antibodies that bind to human activin A but not to activin A from other species. The present invention also includes anti-activin A antibodies that bind to human activin A and activin A from one or more non-human species. For example, the anti-activin A antibodies of the present invention may bind to human activin A and, in some cases, may or may not bind to one or more of the activin A of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus macaque, marmoset, rhesus macaque, or chimpanzee. According to certain exemplary embodiments of the present invention, anti-activin A antibodies that specifically bind to human activin A (e.g., activin A or a βA subunit-containing heterodimer) and cynomolgus macaque (e.g., Macaca fascicularis) activin A are provided.

[0115] multispecific antibodies The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., J Immunol 147:60-69 (1991), Kufer et al., Trends Biotechnol 22:238-244 (2004). The anti-Activin A antibodies of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. For example, the present invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for human Activin A or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or conjugated to a therapeutic moiety. One embodiment of the present invention includes a bispecific antibody in which one arm of the immunoglobulin is specific for human Activin A or a fragment thereof, and the other arm of the immunoglobulin is specific for GDF8.

[0116] Exemplary bispecific antibody formats that can be used in the context of the present invention include the use of a first immunoglobulin (Ig)C H 3 domain and a second IgC H 3 domain, wherein the first and second IgC H 3 domains differ from each other by at least one amino acid, and the difference of at least one amino acid reduces the binding of the bispecific antibody to Protein A as compared to a bispecific antibody lacking this amino acid difference (see, for example, U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety). In one embodiment, the first Ig C H 3 domain binds to Protein A, and the second Ig C HThe three domains contain mutations that reduce or eliminate protein A binding, such as H95R modification (based on IMGT exon numbering, H435R based on EU numbering). Second C H 3 may further include Y96F modification (by IMGT, Y436F by EU). Second C H Further modifications that may be found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT, D356E, L358M, N384S, K392N, V397M, and V422I according to EU), and for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT, E This may further include N384S, K392N, and V422I from U, and, in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, V82I, and L105P (from IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, V422I, and L445P from EU). Variations of the bispecific antibody format described above are considered within the scope of the present invention.

[0117] Other exemplary dual-specificity formats that can be used in the context of the present invention include, for example, scFv system formats or diabody dual-specificity formats, IgG-scFv fusion, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain having knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab(DAF)-IgG, and Mab 2This includes, but is not limited to, the bispecificity format (for a review of the above formats, see, for example, Klein et al., mAbs 4:6, 1-11 (2012) and the references cited therein). Bispecificity antibodies can also be constructed using peptide / nucleic acid conjugations, for example, using non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into a multimeric complex having a defined composition, valence, and geometric shape (see, for example, Kazane et al., J Am Chem Soc. 135(1):340 / 346 (2013)).

[0118] Therapeutic formulations and administration The anti-activin A antibody (or other anti-activin A-specific antagonist) used in the method of the present invention may be formulated for administration in a pharmaceutical composition using one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition is formulated with a suitable carrier, excipient, and other agents that provide improved mobility, delivery, tolerance, etc. Many suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids (cationic or anionic) containing vesicles (LIPOFECTIN®, Life Technologies, Carlsbad, CA, etc.), DNA complexes, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations," PDA, J Pharm Sci Technol 52:238-311 (1998).

[0119] The dose of antibody administered to a patient may vary depending on the patient's age and build, target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the antibody of the present invention is used to treat symptoms or diseases associated with activin A activity in adult patients, it may be advantageous to administer the antibody in a single dose of approximately 0.01 to approximately 20 mg / kg body weight, more preferably approximately 0.02 to approximately 7, approximately 0.03 to approximately 5, or approximately 0.05 to approximately 3 mg / kg body weight. In some embodiments, the dose is 3 mg / kg. In some embodiments, the dose is 10 mg / kg. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Patients with “severe” diseases (e.g., COVID-19) require oxygen supplementation via a nasal cannula, simple face mask, or other similar oxygen delivery device. Patients with the most severe cases of the disease (e.g., COVID-19) require oxygen supplementation delivered via a high-flow nasal cannula non-rebreathing mask, or the use of invasive or non-invasive ventilation, or treatment in an intensive care unit. The effective dose and schedule for administering anti-activin A antibodies can be determined experimentally, for example, by monitoring patient progression through periodic assessments and adjusting the dose accordingly. Furthermore, interspecies scaling of the dose can be performed using methods well known in the art (e.g., Mordenti et al., Pharmaceut Res 8:1351 (1991)).

[0120] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, including, for example, liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or other therapeutic proteins of the present invention, and receptor-mediated endocytosis (see, for example, Wu et al., J Biol Chem 262:4429-4432 (1987)). The antibodies and other therapeutic active ingredients of the present invention can also be delivered by gene therapy techniques. Delivery methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by injection or bolus injection, by absorption through the epithelial or mucocutaneous linings (e.g., oral mucosa, rectum, and intestinal mucosa), and can be administered together with other bioactive agents. Administration may be systemic or topical.

[0121] Pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices facilitate the application of the pharmaceutical compositions of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0122] Numerous reusable pen-type and auto-injector delivery devices have applications for subcutaneous delivery of the pharmaceutical compositions discussed herein. Examples include AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN Examples of disposable pen-type delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, STARLET (trademark) and OPTICLIK (trademark) (sanofi-aventis, Frankfurt, Germany). Examples of, but are not limited to, STARLET (trademark) and OPTICLIK (trademark) (sanofi-aventis), FLEXPEN (trademark) (Novo Nordisk), and KWIKPEN (trademark) (Eli Lilly), SURECLICK (trademark) auto-injector (Amgen, Thousand Oaks, CA), PENLET (trademark) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (trademark) pen (Abbott Labs, Abbott Park, IL).

[0123] In certain circumstances, pharmaceutical compositions can be delivered via a sustained-release system. In one embodiment, a pump can be used (see Langer, Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987)). In another embodiment, a polymer material can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In yet another embodiment, the sustained-release system can be placed near the target of the composition, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, vol.2, pp.115-138). Other sustained-release systems are discussed in the review by Langer, Science 249:1527-1533 (1990).

[0124] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants.

[0125] Combination therapy The present invention includes methods comprising using or administering any of the anti-activin A antibodies described herein in combination with one or more additional therapeutic active ingredients. In some cases, the anti-activin A antibodies of the present invention may also be administered and / or co-formulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, metal chelators, IFN-γ, and / or NSAIDs. In some cases, the anti-activin A antibodies of the present invention may also be administered or used in combination with additional active agents or other supportive therapies to treat, prevent, or reduce the severity of heart failure or one or more complications of heart failure. In some cases, additional active drugs or other supportive therapies include pacemakers, implantable defibrillators, contractility regulators, cardiac resynchronization therapy, ventricular assist devices, biventricular resynchronization therapy, heart transplants, adrenergic blockers (alpha-blockers and beta-blockers), central alpha-agonists, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, positive inotropic agents, vasodilators, benzodiazepines, renin inhibitors, antithrombotic agents, various types of diuretics, captopril, enalapril, lisinopril, benazepril, ramipril, zofenopril, quinapril, perindopril, and lisinopril. Benazepril, imidapril, trandolapril, cilazapril and fosinopril, losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan, fimasartan, propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutrol, pindolol, sotalol, timolol, acebutrol, atenolol, betaxolol, bisoprolol, ceriprolol, esmolol, metoprolol, nebibolol, butaxamine, ICI-118, 551, SR 59230A, phenoxybenzamine, phentolamine, trazoline, trazodone, alfuzosin, doxazosin mesylate (Cardura and Carduran), prazosin, tamsulosin, terazosin, silodosin, atipanmezole (e.g., antisedan),Idazoxane, mirtazapine, yohimbine, acidotic salts (e.g., CaCl2 and NH4Cl), arginine vasopressin receptor 2 antagonists, selective vasopressin V2 antagonists, Na-H exchanger antagonists, carbonate anhydrase inhibitors, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazides, xanthines, dihydropyridines, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, crebidipine, isradipine, efonidipine, felodipine, lasidipine, relcanidipine, Manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, phenylalkylamine calcium channel blockers, verapamil, garopamil, fendiline, benzothiazepine calcium channel blockers, diltiazem, mibefradil, bepridil, flunarizine, fluspirylene, fendiline, gabapentinoids, diconotide, digoxin, amiodarone, berberine, levocimendan, omecamutib, catecholamines, eicosanoids, phosphodiesterase inhibitors, enoximon, milrinone, amlinol Theophylline, glucagon, insulin, sodium nitroferricyanide, hydralazine, isosorbide dinitrate and isosorbide mononitrate, nitroglycerin, benzodiazepines, renin inhibitors, clonidine, guanabenz, guanfacine, methyldopa and moxonidine, minoxidil, guanethidine, mecamylamine, reserpine, irreversible cyclooxygenase inhibitors, adenosine diphosphate receptor inhibitors, clopidogrel, prasugrel, ticagrelol and ticlopidine (phosphodiesterase inhibitors, cilostazol, protease activators) Receptor-1 antagonists, volapaxal, glycoprotein inhibitors, absiximab, eptifivatide, tyrofiban, adenosine reuptake inhibitors, dipyridamole, thromboxane inhibitors, thromboxane synthase inhibitors and thromboxane receptor antagonists, tissue plasminogen activator, alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase, dabigatran, rivaroxaban), apixaban, coumarin, heparin and its derivatives, factor Xa inhibitors, rivaroxaban, apixaban,The antibody is selected from the group consisting of edoxaban, betrixaban, retaxaban, eribaxaban, hirudin, repirudine, bivalirudine, argatroban, dabigatran, ximelagatran, antithrombin protein, batroxobin, hementin, and vitamin E. In some embodiments, any of the anti-activin A antibodies of the present invention may be administered in combination with a GDF8 inhibitor (e.g., an anti-GDF8 antibody) and / or co-formulated.

[0126] Additional therapeutic active ingredients or supportive therapies may be administered to or used on the subject before administration of the anti-activin A antibody of the present invention. For example, if the first component is administered / used one week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration / use of the second component, the first component may be considered administered / used "before" the second component. In other embodiments, additional therapeutic active ingredients or supportive therapies may be administered to or used on the subject after administration of the anti-activin A antibody of the present invention. For example, if the first component is administered / used 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, and 72 hours after the administration / use of the second component, the first component may be considered administered / used "after" the second component. In further embodiments, additional therapeutic active ingredients or supportive therapies may be administered or used to the subject simultaneously with the administration of the anti-activin A antibody of the present invention. "Simultaneous" administration for the purposes of the present invention includes, for example, the administration of the anti-activin A antibody and the additional therapeutic active ingredient to the subject in a single dosage form, or in separate dosage forms administered to the subject within approximately 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (for example, both the anti-activin A antibody and the additional therapeutic active ingredient may be administered intravenously, subcutaneously, intravitreally, etc.). Alternatively, each dosage form may be administered via a different route (for example, anti-activin A antibody may be administered topically (e.g., intravitreally), and additional therapeutic active ingredients may be administered systemically). In any event, administration of this ingredient as a single dose, in different dosage forms via the same route, or in different dosage forms via different routes is considered “concurrent administration” for the purposes of this disclosure. For the purposes of this disclosure, administration of anti-activin A antibody “before,” “at the same time as,” or “after” the administration of additional therapeutic active ingredients is considered administration of anti-activin A antibody “in combination with” the additional therapeutic active ingredient.

[0127] The present invention includes pharmaceutical compositions in which the anti-activin A antibody of the present invention is co-formulated with one or more additional therapeutic active ingredients described elsewhere in this specification.

[0128] Dosage The amount of the active ingredient that can be administered to the target (e.g., an anti-activin A antibody, an anti-GDF8 antibody, or other therapeutic agents given in combination with an anti-activin A antibody, or a bispecific antibody that specifically binds to activin A and GDF8) is generally a therapeutically effective dose, as discussed elsewhere in this specification.

[0129] In some embodiments, the therapeutically effective dose is approximately 0.05 mg to approximately 600 mg, for example, approximately 0.05 mg, approximately 0.1 mg, approximately 1.0 mg, approximately 1.5 mg, approximately 2.0 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg. g, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330m g, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg , about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg , about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg The antibodies can be approximately 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, or 1000 mg.

[0130] The amount of anti-activin A antibody or other therapeutic agent contained in each dose may be expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, anti-activin A, anti-GDF 8, and / or anti-activin A / anti-GDF 8 bispecific antibodies may be administered to a patient in doses ranging from approximately 0.0001 to approximately 50 mg / kg of patient body weight (e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, 10 mg / kg). It can be administered in doses such as 0.0 mg / kg, 10.5 mg / kg, 11.0 mg / kg, 11.5 mg / kg, 12.0 mg / kg, 12.5 mg / kg, 13.0 mg / kg, 13.5 mg / kg, 14.0 mg / kg, 14.5 mg / kg, 15.0 mg / kg, 15.5 mg / kg, 16.0 mg / kg, 16.5 mg / kg, 17.0 mg / kg, 17.5 mg / kg, 18.0 mg / kg, 18.5 mg / kg, 19.0 mg / kg, 19.5 mg / kg, 20.0 mg / kg, etc.

[0131] Administration regimen According to certain embodiments of the present invention, multiple doses of the active ingredient (e.g., a pharmaceutical composition comprising a combination of an anti-activin A antibody and any additional therapeutic activator as referred herein, including an anti-activin A antibody, an anti-GDF8 antibody administered in combination with an anti-activin A antibody, or an anti-activin A antibody and a bispecific antibody that specifically binds to activin A and GDF8) can be administered to a subject over a specified period of time. Methods according to this aspect of the present invention include administering multiple doses of the active ingredient of the present invention to a subject in succession. As used in the present invention, “administering in succession” means that each dose of the active ingredient is administered to the subject at different points in time, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present invention includes methods comprising administering a single initial dose of the active ingredient, followed by one or more secondary doses of the active ingredient, and optionally one or more tertiary doses of the active ingredient, to a patient in succession.

[0132] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of the active ingredient, e.g., the anti-activin A antibody of the present invention, or the combination therapy of the present invention, e.g., the anti-activin A antibody and the anti-GDF8 antibody. Therefore, the “initial dose” is the dose administered at the start of the treatment regimen (also called the “baseline dose”). The “secondary dose” is the dose administered after the initial dose, and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of the active ingredient, e.g., the anti-activin A antibody, but may generally differ from each other in terms of administration frequency. However, in certain embodiments, the amounts of anti-activin A antibody contained in the initial, secondary, and / or tertiary doses may differ from each other during the course of treatment (e.g., increased or decreased as needed). In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered as a “loading dose” at the start of the treatment regimen, followed by subsequent doses (e.g., “maintenance doses”) administered at a lower frequency.

[0133] In certain exemplary embodiments of the present invention, each secondary dose and / or tertiary dose is administered after 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2 or more) weeks after the previous dose. As used herein, the phrase "previous dose" means, in a series of multiple administrations, the dose of the active ingredient, e.g., an anti-activin A antibody, administered to the patient immediately before the administration of the immediately subsequent dose in sequence without intervening doses.

[0134] The method according to this aspect of the invention may include administering to the patient any number of secondary doses and / or tertiary doses of the active ingredient of the invention, e.g., an anti-activin A antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0135] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the most recent dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the most recent dose. In certain embodiments of the present invention, the frequency at which secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

[0136] The present invention includes a dosing regimen in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by 2 or more maintenance doses administered to the patient at a less frequent frequency. For example, according to this aspect of the present invention, if the loading dose is administered once a month, the maintenance dose may be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0137] kit The present invention further provides a product or kit comprising packaging materials, a container, and a pharmaceutical product contained within the container, wherein the pharmaceutical product comprises at least one activin A antagonist (e.g., an anti-activin A antibody), and the packaging materials include a label or accompanying document indicating instructions and information for use (e.g., use of an anti-activin A antibody to treat cardiac dysfunction or heart failure). [Examples]

[0138] The following examples are provided to give a complete disclosure and explanation of how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be the present invention. Although efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some degree of experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0139] Example 1. Generation of human antibodies against activin A An immunogen containing activin A protein (inhibin-βA dimer) was directly administered to VELOCIMMUNE® mice, which contain DNA encoding the human immunoglobulin heavy chain and kappa light chain variable region, along with an adjuvant to stimulate an immune response. The antibody immune response was monitored by an activin A-specific immunoassay. When the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce activin A-specific antibodies. Using this technique, several anti-activin A chimeric antibodies (i.e., antibodies having a human variable domain and a mouse constant domain) were obtained. An exemplary antibody obtained in this way is H2aM10965N. Subsequently, the human variable domain derived from the chimeric antibody was cloned onto the human constant domain to produce the fully human anti-activin A antibody described herein.

[0140] Furthermore, as described in U.S. Patent Application Publication No. 2007 / 0280945(A1), several anti-activin A antibodies were isolated directly from antigen-positive B cells without fusing them to myeloma cells. Using this method, several fully human anti-activin A antibodies (i.e., antibodies having a human variable domain and a human constant domain) were obtained. The exemplary antibodies thus produced were named as follows: H4H10423P, H4H10429P, H4H10430P, H4H10432P2, H4H10440P2, H4H10442P2, H4H10436P2, and H4H10446P2.

[0141] The specific biological properties of exemplary anti-activin A antibodies produced according to the method of this embodiment will be described in detail in the following examples.

[0142] Example 2. Amino acid sequences of the heavy chain variable region and the light chain variable region. Table 1 lists the amino acid sequence pairs of the heavy chain variable region and light chain variable region of the antibodies specific to the selected anti-activin A antibodies and their corresponding antibody identifiers. The corresponding nucleic acid sequence identifiers are shown in Table 2.

[0143] [Table 1]

[0144] [Table 2]

[0145] Antibodies are typically described herein according to the following nomenclature: an Fc prefix (e.g., "H1M", "H2aM", "H4H"), followed by a numerical identifier (e.g., "10423", "10424", or "10426", as shown in Tables 1 and 2), followed by the suffix "P", "P2", or "N". Therefore, according to this nomenclature, antibodies may be referred herein, for example, to "H4H10423P", "H4H10432P2", "H2aM10965N", etc. The H1M, H2M, and H4H prefixes in antibody names used herein indicate a specific Fc region isotype of the antibody. For example, an "H2aM" antibody has mouse IgG2a Fc, while an "H4H" antibody has human IgG4 Fc. As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (for example, an antibody having mouse IgG2a Fc can be converted to an antibody having human IgG4), but in either case, the variable domain (including the CDR) indicated by the numerical identifier shown in Table 1 remains the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0146] Control structures used in the following examples Anti-activin A control molecules were included in the following examples for comparative purposes. The control antibody referred to herein as Control 1 is a human anti-activin A antibody having the heavy and light chain variable domain sequences of "A1" as described in U.S. Patent No. 8,309,082. Control 2 is an anti-human activin receptor type II B antibody (anti-ActR2B mAb) disclosed as MOR8159 in U.S. Patent Application No. 2012 / 0237521(A1). Control 3 is a mouse anti-activin A monoclonal antibody (catalog number MAB3381) purchased from R&D Systems (Minneapolis, MN). Control 4 is an activin IIB receptor-Fc fusion molecule (a soluble activin RIIB receptor extracellular domain produced using a C-terminal human IgG1 Fc fusion protein (NP_001097 E23-P133, followed by a Gly-Ser linker, followed by a C-terminal human IgG1 Fc fusion)), the sequence of which is shown as SEQ ID NO: 227.

[0147] Example 3. Antibody binding to human activin A determined by surface plasmon resonance. Real-time surface plasmon resonance biosensors (Biacore T200 or Biacore 4000, GE Healthcare Life Sciences, Piscataway, NJ) assays were used at 25°C and 37°C to measure the binding affinity and rate constant of antigen binding to selected purified anti-human activin A monoclonal antibodies. Antibodies expressed as either mouse Fc (prefixed H2aM) or human Fc (prefixed H4H) were captured on the surface of their respective anti-Fc sensors (mAb capture format). Goat anti-mouse IgG polyclonal antibody (GE Healthcare, #BR-1008-38) or mouse anti-human IgG monoclonal antibody (GE Healthcare) were prepared by direct amine coupling to Biacore CM5 sensor chips. Anti-activin A antibodies were captured on one of the surfaces of Healthcare (#BR-1008-39). Dynamics experiments were performed using either HBS-EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20, pH 7.4) or PBS-P (10mM sodium phosphate, 2.7mM KCl, 137mM NaCl, 0.02% NaN3, 0.05% Surfactant P20, pH 7.4) as both the running buffer and sample buffer. Various concentrations (4-fold dilutions ranging from 50 to 0.2nM) of activin A (R&D Systems, #338-AC-050 / CF), activin B (R&D Systems, #659-AB-025 / CF), activin AB (R&D Systems, #1006-AB-005), and activin AC (R&D Systems) were used. The antigen-antibody association rate was measured by injecting either Systems (#4879-AC / CF) or inhibin E (Novus Biologicals, #H00083729-P01) onto the surface of the captured antibody. Antibody-antigen association was monitored for 240 seconds, while dissociation in buffer was monitored for 600 seconds.The rate constants for dynamic association and dissociation were determined by processing and fitting the data using Scrubber software version 2.0c. The binding equilibrium dissociation constant (K) was then determined. D ) and dissociation half-life (t 1 / 2 ) was calculated from the dynamic speed constant as follows: K D (M=k d / k a and t 1 / 2 (minutes) = [ln2 / (60 * k d Tables 3-10 show the dynamic binding parameters of different anti-activin A monoclonal antibodies. (NB = no binding observed under the conditions used; NT = not tested).

[0148] [Table 3]

[0149] Regarding the kd values ​​in italics, no dissociation of the analyte was observed under these experimental conditions, and therefore the kd value is 5.0E-05 s. -1 It was fixed in place.

[0150] [Table 4]

[0151] Regarding the kd values ​​in italics, no dissociation of the analyte was observed under these experimental conditions, and therefore the kd value is 5.0E-05 s. -1 It was fixed in place.

[0152] [Table 5]

[0153] [Table 6]

[0154] [Table 7]

[0155] [Table 8]

[0156] [Table 9]

[0157] [Table 10]

[0158] As shown in Tables 3 and 4, the anti-activin A antibody of the present invention has a K content ranging from less than 3.18 pM (i.e., ≤3.18E-12) to 745 pM (i.e., 7.45E-10) at 25°C. D The value, and the range of K at 37°C from less than 2.18 pM (i.e., ≤2.18E-12) to 1.77 nM (1.77E-09). D Activin A bound to the antibody was measured at a value. As shown in Tables 5 and 6, some anti-activin A antibodies (i.e., H4H10432P2, H4H10442P2, H4H10430P2, H4H10446P2, and H4H10468P2) did not show measurable binding to activin B at 25°C or 37°C. Some antibodies showed K levels in the range of approximately 18.5 pM (i.e., 1.85E-11) to 33.1 nM (i.e., 3.31E-08) at 25°C (Table 7) and approximately 44.3 pM (i.e., 4.43E-11) to 24.2 nM (i.e., 2.42E-08) at 37°C (Table 8). D The values ​​indicated measurable binding to activin AB. Some antibodies showed K levels ranging from approximately 99.7 pM (i.e., 9.97E-11) to 11.8 nM (i.e., 1.18E-08) at 25°C (Table 9) and from approximately 462 pM (i.e., 4.62E-10) to 22.5 nM (i.e., 2.25E-08) at 37°C (Table 10). DThe values ​​indicated measurable binding to activin AC. Furthermore, none of the anti-activin A antibodies of the present invention tested showed measurable binding to inhibin E (data not shown).

[0159] Example 4. Inhibition of activin A-mediated receptor activation and SMAD complex signaling by an anti-activin A antibody. To further characterize the anti-activin A antibodies discussed herein, bioassays were developed to detect the activation of activin type IIA and activin type IIB receptors (ActRIIA and ActRIIB, respectively), as well as the subsequent phosphorylation and activation of the activin type I receptor. The interaction between ActRIIA and ActRIIB and activin leads to the induction of diverse cellular processes, including proliferation regulation, cancer cell metastasis, and embryonic stem cell differentiation (Tsuchida, K. et al., Cell Commun Signal 7:15 (2009)). Phosphorylation and activation of the type I receptor result in phosphorylation of SMAD 2 and 3 proteins, which form the activated SMAD complex, leading to gene transcriptional regulation.

[0160] To detect activation of the SMAD complex signaling pathway via activin binding to the activin type II receptor, human A204 rhabdomyosarcoma cell line (ATCC, #HTB-82) was transfected with the Smad 2 / 3-luciferase reporter plasmid (CAGAx12-Luc; Dennler, 1998) to create the A204 / CAGAx12-Luc cell line. The A204 / CAGAx12-Luc cells were maintained in McCoy's 5A (Irvine Scientific, #9090) supplemented with 10% fetal bovine serum (FBS), penicillin / streptomycin / glutamine, and 250 μg / mL of G418. For the bioassay, A204 / CAGAx12-Luc cells were seeded at 10,000 cells / well on 96-well assay plates in low-serum medium, 0.5% FBS, and OPTIMEM (Invitrogen, #31985-070), and incubated overnight at 37°C and 5% CO2. To determine the ligand dose response, activin A (R&D Systems, #338-AC), activin B (R&D Systems, #659-AB), activin AB (R&D Systems, #1066-AB), and activin AC (R&D Systems, #4879-AC / CF) were added to the cells in serial dilutions of 1:3 from 100 to 0.002 nM, and a control without activin was also provided. Activin A, Activin B, Activin AB, and Activin AC have EC values ​​of 99 pM, 47 pM, 19 pM, and 4.4 nM, respectively. 50The antibody was observed to activate the A204 / CAGAx12-Luc cell line. To measure inhibition, the antibody was serially diluted 1:3 to 100–0.002 nM, 1000–0.02 nM, or 300–0.005 nM (including control samples containing appropriate isotype control antibodies or control samples without antibodies), and 100 pM activin A, 50 pM activin B, 30 pM activin AB, or 4 nM activin AC were added to the cells at a constant concentration. Control 4 (ActRIIB-hFc; SEQ ID NO: 227) was used as a positive blocking control in this assay. After incubation at 37°C and 5% CO2 for 5.5 hours, OneGlo substrate (Promega, #E6051) was added, and luciferase activity was detected using a Victor X (Perkin Elmer) instrument. The results were analyzed using nonlinear regression (4-parameter logistic regression) with Prism 5 software (GraphPad).

[0161] As shown in Table 11, the anti-activin A antibody of the present invention has an IC in the range of 39 pM to 3.5 nM. 50 Activin A at 100 pM was blocked, while control 1 blocked IC at 83 pM. 50 Blockage was achieved based on the value. A subset of the anti-activin A antibodies of the present invention were tested for blockade of activin B, AB, and AC. Of the nine antibodies tested, four had IC values ​​in the range of 130 pM to 100 nM. 50 The antibody blocked activin B at a concentration of 50 pM. The five antibodies of the present invention tested for activin B blockade blocked only at high antibody concentrations, while control 1 showed no measurable activin B blockade. The eight antibodies of the present invention tested showed IC50 in the range of 100 pM to 8.2 nM. 50 The activin AB at 30 pM was blocked, while control 4 blocked IC at 540 pM. 50 Blockage was observed based on the value. Only one antibody, H4H10423P, showed weak blockage of activin AB. Of the eight antibodies tested, seven showed IC values ​​in the range of 580 pM to 6.5 nM. 50 The activin AC of 4nM was blocked by the value, while control 4 blocked the IC of 1.1nM. 50Blockage was observed based on the value. One antibody, H4H10423P, did not demonstrate any blockage of activin AC. Neither mouse IgG (mIgG isotype control) nor human IgG (hIgG isotype control) negative controls blocked receptor ligand activation.

[0162] [Table 11]

[0163] The bioassay using A204 / CAGAx12-Luc cells could also be stimulated with GDF8 (R&D Systems, catalog no. 788-G8 / CF) and GDF11 (R&D Systems, catalog no. 1958-GD-010 / CF). The above conditions were used to test the functional inhibition of these ligands by activin A antibodies, but the assay was performed by replacing the activating ligand with GDF8 or GDF11, yielding EC50 values ​​of 188 pM and 84 pM, respectively. In this assay, activation by a constant concentration of 0.50 nM GDF8 or 0.40 nM GDF11 was completely blocked by control 4, and IC50 was obtained. 50 The values ​​were 298 pM and 214 pM, respectively. When tested with antibodies up to 100 nM using these same constant concentrations of ligands, no inhibition of GDF8 or GDF11 by activin A antibodies, H4H10446P2 and H4H10430P, was observed. On a different day, activin A antibodies H4H10429P and H4H10436P2 were tested for inhibition in this assay in the presence of constant concentrations of 250 pM GDF8 or 250 pM GDF11, and no inhibition was observed after incubating cells with test activin A antibodies up to 150 nM. GDF8 and GDF11 alone in this assay showed EC50 values ​​of 124 pM and 166 pM, respectively. These data demonstrate that the activin A antibodies H4H10446P2, H4H10430P, H4H10429P, and H4H10436P2 do not functionally inhibit GDF8 or GDF11.

[0164] Example 5. Blocking of activin A binding using an activin A antibody. The ability of selected anti-activin A antibodies to block interactions between activin A and its receptors, ActRIIB and ActRIIA, as well as its endogenous antagonist, follistatin, was measured using the Biacore 3000 instrument. In this experiment, control 4 (human ActRIIB co-expressed with a C-terminal human Fc tag (SEQ ID NO: 227)), human ActRIIA co-expressed with a C-terminal human Fc tag (hActRIIA-Fc; R&D Systems, #340-R2-100), or follistatin-288 (R&D Systems, #5836-FS-025) was amine-coupled to the surface of the Biacore CM5 sensor. Fixed-concentration 5 nM activin A (R&D Systems, #338-AC) was incubated alone or mixed with activin A antibody, hActRIIA-Fc, hActRIIB-Fc, or isotype control antibody at a final concentration of 60 nM (12-fold molar excess relative to activin A) for 1 hour at room temperature. The antibody-activin A mixture was then injected at a flow rate of 20 uL / min onto amine coupling control 4, hActRIIA-Fc, or follistatin-288 surfaces. The binding signal (RU) was measured 150 seconds after the start of injection, and the specific binding signal was determined by subtracting this signal from the RU value measured for the negative control reference surface. The percentage of free activin A binding to the receptor or antagonist surface in the presence of each anti-activin A antibody was calculated as the ratio of the observed specific binding signal to the specific binding signal from 5 nM activin A in the absence of the antibody.

[0165] [Table 12]

[0166] As shown in Table 12, six of the seven anti-activin A antibodies tested, as well as both control 1 and control 3, blocked the binding of actin A to follistatin-288. One antibody, H4H10423P, did not prevent the binding of activin A to follistatin-288. Control 4 and hActRIIA-Fc blocked the binding of activin A to follistatin-288 at higher concentrations.

[0167] [Table 13]

[0168] As shown in Table 13, four of the seven anti-activin A antibodies tested, as well as both control 1 and control 3, blocked the binding of hActRIIA-Fc to activin A. Three antibodies, H4H10442P2, H4H10446P2, and H4H10423P, did not prevent the binding of activin A to hActRIIA-Fc. Control 4 and hActRIIA-Fc blocked the binding of activin A to hActRIIA-Fc.

[0169] [Table 14]

[0170] As shown in Table 14, four of the seven anti-activin A antibodies tested, as well as both control 1 and control 3, blocked the binding of activin A to hActRIIB-Fc. Two antibodies, H4H10442P2 and H4H10446P2, did not block the binding of activin A to hActRIIB-Fc. One antibody, H4H10423P, showed the ability to partially block the binding of activin A to hActRIIB-Fc at higher concentrations than the antibodies tested. Both hActRIIB-Fc and hActRIIA-Fc blocked the binding of activin A to hActRIIB-Fc.

[0171] Example 6. Activin A induces upstream signaling and activates cardiac stress genes in human induced pluripotent stem cell-derived cardiomyocytes. For culturing human induced pluripotent stem cell-derived (IPSC) cardiomyocytes, tissue culture vessels were pre-coated with 10 μg / mL fibronectin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 1 hour. 2 Cellular Dynamics (Fujifilm Cellular Dynamics, Madison, WI, USA) was stored, thawed, and seeded according to the manufacturer's instructions. Briefly, the cells were rapidly thawed (37°C, 3 minutes) and slowly diluted with seeding medium. For gene expression analysis and phosphorylation assays, 5 × 10⁶ cells were used per well in a 12-well plate. 5 Individual cells were seeded. For impedance, electrophysiology, and calcium flux assays, cells were placed in wells of 5 × 10⁶ cells each. 4 Cells were seeded at a specific cell density into 96-well plates. The cells were maintained in a humidified 37°C incubator containing 5% CO2, with the culture medium changed every 48 hours. Before starting each experiment, the cells were maintained in culture medium until a synchronized pulsating monolayer of cells was formed (approximately 10–14 days).

[0172] To detect SMAD phosphorylation, cells were exposed to 1 nM activin A (R&D Systems, Minneapolis, MD, USA) for 30 minutes. Cells were washed twice with chilled phosphate-buffered saline and lysed using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) supplemented with Halt® Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The lysates were centrifuged (14,000 × g, 15 min), and total protein was quantified using the Pierce BCA Protein Quantification Kit (Thermo Fisher Scientific). Protein detection in cell lysates was performed under reducing conditions using the 12-230 kDa Separation Module for capillary electrophoresis Wes® System (ProteinSimple, San Jose, CA, USA) according to the manufacturer's instructions. Protein samples were diluted to a final concentration of 0.5 mg / mL with 5 × reducing buffer, denatured (5 min, 95°C), and placed on ice. Cartridge plates were assembled, centrifuged (1000×g, 5 minutes), and placed in a Wes™ instrument. Primary antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA). Phospho-SMAD 2 (Ser465 / 467) / SMAD3 (Ser423 / 425) was diluted 1:50, SMAD2 / 3 was diluted 1:50, and GAPDH was diluted 1:100. The anti-rabbit detection module (ProteinSimple) was supplied with antibody diluent, 1× anti-rabbit secondary antibody, streptavidin-HRP conjugate, and chemiluminescence detection reagent. Protein detection was analyzed using Compass software (ProteinSimple), and the area under the curve and height of the peak chemiluminescence signal from the target protein were quantified. SMAD2 / 3 phosphorylation was significantly increased by 83% in iPSC-CMs exposed to 1 nM activin A for 30 minutes (P<0.001). The inhibitory activin A antibody (mAb2; H$H10446P2) blocked this increase in SMAD phosphorylation (see Figure 12).These data demonstrate that activin A induces signal transduction in iPSC-CMs.

[0173] For gene expression analysis, a PCR reaction (total 20 μL) containing 10 μL of 2× TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), 1 μL of 20× TaqMan probe, 5 μL (10 ng) of cDNA, and 4 μL of water was performed on a QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). The thermocycler settings were as follows: 95°C for 15 minutes, followed by 40 cycles of 15 seconds at 95°C, and then 60°C for 60 seconds. Amplification plots were created using QuantStudio® 3 instrument software, and the resulting cycle threshold (Ct) values ​​were derived. GAPDH was used as an endogenous control. For all RT-qPCR analyses, the delta-delta Ct(2) ratio was calculated. -ΔΔCt The relative scalar changes in gene expression were calculated using the Pfaffl 2001 method. Exposure of IPSC cardiomyocytes to activin A (acute - 24 hours, or chronic - 6 treatments) activated the expression of the downstream activin A signaling genes FSTL3 (also known as FLRG) and serpin 1 (also known as PAI-1), as well as atrial natriuretic peptide (NPPA) and type B natriuretic peptide (NPPB), which are common markers of cardiac stress (see Figure 10).

[0174] Example 7. Anti-activin A induces contractile dysfunction and electrophysiological dysfunction of IPSC-cardiocardial cells blocked by anti-activin A antibodies. The contractility (impedance) and electrophysiology of iPSC-CMs were characterized using the CardioExcyte 96 (Nanion Technologies, Munich, Germany), a hybrid system capable of simultaneously recording the impedance (contractility) and extracellular field potential (EFP) of the beating monolayer of cardiomyocytes in a label-free environment under physiological culture conditions. In this study, iPSC-CMs were seeded in 96-well plates containing electrodes (NSP-96; Nanion Technologies), and recordings were made for 30 seconds every 4 hours. Impedance and EFP data were analyzed using Data Control 96 software (Nanion Technologies).

[0175] IPSC cardiomyocytes were seeded, and their contraction amplitude (impedance) was measured throughout the experiment while they were exposed to culture medium or various concentrations of activin A (R&D Systems) once or continuously. As shown in Figure 1B, long-term exposure of IPSC cardiomyocytes to activin A (6 treatments) resulted in a dose-dependent trend of decreasing amplitude. Minimal effects were observed with single exposures (see Figure 1A).

[0176] As described above, using seeded human iPSC cardiomyocytes, the anti-activin A antibody (mAb1; H4H10430P) prevented dysfunction. As shown in Figure 2, application of mAb1 prevented a decrease in systolic amplitude across all doses tested compared to isotype controls.

[0177] For EFP recording, transient extracellular electrical activity was measured, followed by reversing the mean beat to mimic the action potentials of cardiomyocytes. Amplitude, downstroke velocity (maximum gradient during depolarization), and electric field potential duration (FPDMax: time between the initial deflection of depolarization and the maximum of the repolarization curve) were characterized for each mean beat. Compared to controls, cardiomyocytes chronically treated with activin A (1 nM) showed elongated action potentials (0.56 ± 0.01 vs. 0.49 ± 0.02 sec, P < 0.01). A 25 nM inhibitory antibody (mAb1) prevented this increase in action potential duration, while an isotype control antibody did not (0.51 ± 0.05 vs. 0.56 ± 0.02 sec). Chronic exposure to activin A caused a decrease in electric field potential amplitude compared to the medium control (48.58±6.52 vs. 74.52±11.66 μV, P<0.01). A 25 nM inhibitory antibody (mAb1) prevented this decrease compared to a 25 nM isotype control antibody (85.55±19.82 vs. 42.87±2.00 μV). Exposure to activin A also reduced the electric field potential downstroke rate compared to the medium control (0.018±0.006 vs. 0.034±0.005 V / sec, P<0.001). The inhibitory antibody (mAb1) of activin A + 25 nM prevented this decrease in downstroke rate, while the 25 nM isotype control antibody did not (0.039 ± 0.009 vs. 0.019 ± 0.002 V / sec) (see Figures 13A and 13B).

[0178] Calcium flux was evaluated using the EarlyTox cardiotoxicity kit (Molecular Devices, San Jose, CA, USA). Calcium dye loading was performed according to the manufacturer's instructions. The EarlyTox calcium dye was resuspended in the supplied buffer and added to cells in a 1:1 ratio with myocardial maintenance medium. After incubating the plates for 2 hours (37°C, 5% CO2), the calcium flux was recorded for 2 minutes at 37°C using the FLIPR Tetra System (Molecular Devices) with the following parameters: excitation: 470–495 nM, emission: 515–575 nM, exposure time: 50 ms, LED intensity: 50%, interval time: 0.1 sec. Traces of the calcium flux were obtained and analyzed using SoftMax Pro Software (Molecular Devices). Chronic treatment with activin A (1 nM) reduced peak calcium flux amplitude (447 ± 33 vs. 609 ± 99 RFU, p < 0.05), increased calcium flux decline time (0.70 ± 0.04 sec vs. 0.52 ± 0.05 sec, p < 0.0001), and increased calcium flux rise time (0.40 ± 0.06 vs. 0.24 ± 0.03 sec, p < 0.01) compared to the medium control. The peak calcium treatment amplitude was 759 ± 129 RFU in cells treated with activin A + inhibitor antibody (mAb1) compared to 484 ± 37 RFU in cells treated with activin A + isotype control antibody. Compared to isotype control antibodies, the activin A inhibitor antibody (mAb1) prevented an increase in calcium flux decline time (0.58±0.04 vs. 0.75±0.08 seconds) and rise time (0.27±0.1 vs. 0.36±0.05 seconds), while the isotype control antibody did not (see Figures 14A and 14B).

[0179] These data suggest that high levels of activin A can directly act on cardiomyocytes and may contribute to heart failure and cardiac dysfunction in the elderly population.

[0180] Example 8. Serum levels of activin A, follistatin-related gene (FLRG), and plasminogen activator inhibitor-1 (PAI-1) were elevated in COVID-19 patients and correlated with disease severity. Serum samples were collected from COVID-19 patients and performed ELISA according to a standard protocol (R&D Systems) to measure the concentrations of activin A, FLRG, and PAI-1. Activin A ELISA samples were diluted 1:1, and FLRG ELISA samples were diluted 1:5. Patient samples were thawed on ice for 1 day, and each sample was divided equally into three ELISAs upon thawing to prevent freeze-thaw effects. A plate control containing male and female serum was prepared using commercially available control serum, and this was used to normalize the data. ELISAs were read using a BioTek Synergy Neo2 Multi-Mode Reader. For FLRG data, if any sample exceeded the standard curve, the result was entered at the maximum volume of the standard curve (4000 pg / mL).

[0181] Statistical data classified by disease severity and oxygen needs were expressed as median (interquartile range: IQR) for continuous variables and as frequency (percent: %) for categorical variables. The Kruskal-Wallis test was used for continuous variables across three or more groups, and the Wilcoxon signed-rank test was used for continuous variables between two groups. Fisher's exact test was used for categorical variables. Differences in activin A, FLRG, and PAI-1 based on baseline disease severity and oxygen needs were tested using the Kruskal-Wallis test with follow-up Dunn pairwise comparisons (for significant omnibus results). For long-term outcomes, including mortality and clinical score improvement (≥1 point), logistic regression models were used with baseline activin A, FLRG, and PAI-1 (standardized) as predictors, with and without covariates. Fine-Gray partial distribution hazard models were also generated for these long-term outcome variables using available time-to-event information. Participants were divided into low and high viral load groups based on the median split of baseline values ​​for each individual analyte. Subdistribution hazard ratios (sHRs) for the high load group relative to the low load group were calculated with and without covariates. All-cause mortality and time to clinical score improvement data were censored at days 60 and 29, respectively. The incidence of each outcome during the study was calculated at the censoring point.

[0182] The results are shown in Figures 3-9, which illustrate significant increases in serum levels of activin A, FLRG, and PAI-1 in COVID-19 patients compared to controls, as well as the correlation between serum levels of activin A, FLRG, and PAI-1 and disease severity. Both FLRG and PAI-1 are biomarkers of activin A pathway activation.

[0183] Activin A and FLRG levels were highest in ICU patients and therefore correlated with the most severely affected COVID-19 patients (Figure 4). The relationship between oxygen needs at study enrollment and all-cause mortality was examined using baseline levels of activin A, FLRG, and PAI-1 in COVID-19 patients. Baseline activin A levels were significantly lower in surviving patients (median = 336.8 pg / mL) than in deceased patients (median = 547.5 pg / mL) (p<0.0001). A similar trend was observed for FLRG, which was significantly lower in surviving patients (median = 12141.8 pg / mL) than in deceased patients (median = 17633.6 pg / mL) (p<0.0001). Baseline PAI-1 levels did not differ significantly by mortality (p=0.52).

[0184] Baseline oxygen administration status was stratified into three categories based on oxygen device type: low flow, high flow, and invasive ventilation (IMV). Differences among these three groups were observed for activin A (H(2)=48.2; p<0.0001), FLRG (H(2)=37.1; p<0.0001), and PAI-1 (H(2)=11.3; p=0.0004). Activin A was lowest in low-flow patients (median = 236.5 pg / mL), higher in high-flow patients (403.7 pg / mL), and highest in IMV patients (median = 499.6 pg / mL). However, activin A did not differ significantly between high-flow and IMV patients (z=1.8, p>0.0167). A similar trend was observed for FLRG, which was lowest in low-flow patients (median = 9399.0 pg / mL), higher in high-flow patients (median = 14117.1 pg / mL), and highest in IMV patients (median = 15424.3 pg / mL). All pairwise comparisons were significant (p<0.0167). PAI-1 was also lower in low-flow patients (median = 16.7 ng / mL) compared to high-flow patients (median = 17.5 ng / mL) and IMV patients (median = 19.2 ng / mL). However, PAI-1 did not differ significantly between low-flow and high-flow patients (z=2.1, p>0.0167), in addition to high-flow and IMV patients (z=1.6, p>0.0167). These data indicate that activin A and its pathway marker, FLRG, correlate with the need for higher oxygen levels, but PAI-1 concentrations, despite being higher in all COVID-19 groups, do not predict oxygen needs. A summary of laboratory results, study progress, and clinical outcomes for patients grouped by baseline oxygen supplementation needs is shown in Table 15 below.

[0185] [Table 15] Note: In statistical analysis, continuous variables are expressed as the median (IQR), and categorical variables are expressed as the count (%).

[0186] As shown in Table 15, for COVID-19 patients, activin A and FLRG (not other pathway markers such as PAI-1) are predictors of the worst-case outcomes of COVID-19, including the need for more invasive oxygen therapy, longer hospital stays, and a higher likelihood of death.

[0187] Considering the unique correlation between activin A and its pathway marker FLRG in COVID-19 patients and the need for oxygen therapy and the risk of death, we investigated the mechanism by which inflammatory cytokines induce activin A.

[0188] Cook Myosite Human Skeletal Muscle derived cells (SKMDCs) were differentiated for 5 days and then co-treated with 100 ng / ml of IL1b or TNFα and activin A induction. Following this, each of the following additional treatments was performed: DMSO (containing only the vehicle as a negative control), IKKi (a downstream Iκ kinase inhibitor; 3 μM withaferrin A), p38i (a p38 inhibitor; 0.3 μM SB203580), JNKi (a JNK inhibitor; 30 μM SP600125), a combination of IKKi + JNKi, or a combination of p38i + JNKi for 24 hours. Activin A concentrations in the acclimatization medium were quantified by ELISA. Within each major treatment condition (IL1b, TNFα), activin A induction for each co-treatment was compared with DMSO co-treatment using pairwise assays. The results are shown in Figure 11. Only significant (p<0.05) Bonferroni-adjusted comparisons are shown. Within the group, each point is a technical replica. Compared to DMSO treatment, activin A induction after IL-1 or TNFα treatment was significantly lower in IKKi-treated cells (t(2)=41.4, p=0.0006). In contrast, cells treated with either JNKi or p38i+JNKi showed far less inhibition of activin A induction compared to DMSO treatment. Combination therapy with IKKi resulted in similar inhibition to IKKi monotherapy. Therefore, IL-1 and TNF induce activin A via the IKK / NF-κB pathway, independently of p38 or JNKi, and increased cytokine levels (e.g., IL-1 and TNFα) correlate with increased oxygen supplementation needs and a higher risk of death in COVID-19 patients.

[0189] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to activin A, for use in a method for treating cardiac dysfunction in a subject requiring such treatment, wherein the method comprises administering the antibody or antigen-binding fragment to the subject, the antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) comprising three complementarity-determining regions comprising amino acid sequences of SEQ ID NOs. 164, 166, and 168, respectively, HCDR1, HCDR2, and HCDR3, and a light chain variable region (LCVR) comprising three complementarity-determining regions comprising amino acid sequences of SEQ ID NOs. 148, 150, and 152, respectively, LCDR1, LCDR2, and LCDR3.

2. The pharmaceutical composition according to claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 162, and the LCVR comprises the amino acid sequence of SEQ ID NO:

146.

3. The pharmaceutical composition according to claim 2, wherein the antibody or its antigen-binding fragment is an antibody containing an IgG heavy chain constant region.

4. The pharmaceutical composition according to claim 3, wherein the IgG heavy chain constant region is an IgG1 isotype.

5. The pharmaceutical composition according to claim 3, wherein the IgG heavy chain constant region is an IgG4 isotype.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is administered to the subject in combination with a GDF8 antagonist.

7. The pharmaceutical composition according to claim 6, wherein the GDF8 antagonist is selected from the group consisting of a GDF8 inhibitory fusion protein, an anti-GDF8 antibody, and an antigen-binding fragment of an anti-GDF8 antibody.

8. The pharmaceutical composition according to claim 7, wherein the GDF8 antagonist is an anti-GDF8 antibody or an antigen-binding fragment thereof.

9. The pharmaceutical composition according to claim 8, wherein the anti-GDF8 antibody or its antigen-binding fragment each comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain containing the amino acid sequence of SEQ ID NO: 218-219-220-222-223-224.

10. The pharmaceutical composition according to claim 9, wherein the anti-GDF8 antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO: 217 and an LCVR containing the amino acid sequence of SEQ ID NO: 221.

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