CN117062609A - Methods for treating or preventing acute respiratory distress syndrome - Google Patents

Abstract

The present disclosure relates to methods of treating or preventing Acute Respiratory Distress Syndrome (ARDS) using compounds that bind to CD131 and neutralize signaling through IL-3, IL-5 and GM-CSF. The disclosure also relates to compounds for use in the treatment or prevention of ARDS and the use of such compounds in the manufacture of a medicament for the treatment or prevention of ARDS.

Description

Methods for treating or preventing acute respiratory distress syndrome

Data of related applications

The present application claims priority from australian patent application No. 2020904755 entitled "method for treating or preventing acute respiratory distress syndrome (Methods for treating or preventing acute respiratory distress syndrome)" filed on month 12 and 21 of 2020 and australian patent application No. 2021903362 entitled "method for treating or preventing acute respiratory distress syndrome (Methods for treating or preventing acute respiratory distress syndrome)" filed on month 10 and 20 of 2021. The entire contents of both applications are hereby incorporated by reference.

Sequence listing

The present application is presented with a sequence listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference.

Technical Field

The present disclosure relates to methods of treating or preventing acute respiratory distress syndrome.

Background

Acute Respiratory Distress Syndrome (ARDS) is a serious complication of several systemic disorders and direct injury to the lungs and is often life threatening. It is associated with high mortality, mainly as a result of multiple organ failure. ARDS occurs when fluid accumulates in the alveoli of the lungs, resulting in less oxygen reaching the blood stream, which deprives the organ of oxygen required for function. Symptoms of ARDS include severe shortness of breath, dyspnea and abnormal shortness of breath, hypotension, and mental confusion and extreme fatigue, which typically occur within hours to days after the initial illness or trauma.

Despite decades of research and advances in medical technology, ARDS-related mortality is still high and none of the drug therapies effectively improve its clinical course. For example, drug candidates that fail in large scale assays include at least glucocorticoids, alprostadil, surfactants, ketoconazole (ketoconazole), N-acetylcysteine, orthocystine, risoproteine and site-inactivating recombinant factor VIIa. Current standard of care is limited to supportive therapies such as oxygenation, mechanical ventilation, fluid management, and prone position.

Thus, there remains a need for new interventions for the treatment and prevention of ARDS.

Disclosure of Invention

In the course of generating the present invention, the inventors identified CD131 (beta co-receptor) as a potential target for pharmaceutical intervention for ARDS. The inventors found that antibodies that bind to CD131 and inhibit the signaling of Interleukin (IL) 3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) successfully reduced several measures of pulmonary inflammation in animal models of ARDS. These findings provide a basis for methods of treating or preventing ARDS in a subject by administering compounds that bind to CD131 and/or inhibit signaling of IL-3, IL-5 and GM-CSF.

Thus, in one embodiment, the present disclosure provides a method for treating or preventing ARDS in a subject comprising administering to the subject one or more compounds that neutralize signaling through IL-3, IL-5 and GM-CSF. Similarly, the present disclosure provides one or more compounds that neutralize signaling through IL-3, IL-5 and GM-CSF for use in treating or preventing ARDS in a subject. The present disclosure also provides the use of one or more compounds that neutralize signaling through IL-3, IL-5 and GM-CSF in the manufacture of a medicament for treating or preventing ARDS.

In one embodiment, the compound binds to one or more of the following:

·IL-5；

·IL-3；

·GM-CSF；

IL-5 receptor alpha (IL-5Ralpha);

IL-3 receptor alpha (IL-3 Ralpha); and

GM-CSF receptor alpha (GM-CSFR alpha).

For example, the methods can include administering a compound, e.g., an antibody that binds to IL-3 and neutralizes signaling through IL-3; and compounds, such as antibodies that bind to GM-CSF and neutralize signaling through GM-CSF; and compounds, such as antibodies that bind to IL-5 and neutralize signaling through IL-5. Likewise, the methods can include administering a compound, such as an antibody that binds to IL-3 ra and neutralizes signaling through IL-3; and compounds, such as antibodies that bind to GM-CSFR alpha and neutralize signaling through GM-CSF; and compounds, such as antibodies that bind to IL-5Rα and neutralize signaling through IL-5. The method may alternatively comprise administering a combination of compounds, wherein one or more compounds bind to one or more of the foregoing cytokines and one or more compounds bind to one or more of the foregoing receptors.

In another embodiment, the method comprises administering a multispecific compound, e.g., an antibody. For example, the multispecific compound is trispecific and binds to IL-3 or IL-3Rα and inhibits signaling through IL-3, and binds to GM-CSF or GM-CSFR α and inhibits signaling through GM-CSF, and binds to IL-5 or IL-5Rα and inhibits signaling through IL-5.

In another embodiment, the method comprises administering a bispecific molecule, e.g. an antibody, and a monospecific compound, e.g. an antibody. For example, bispecific molecules bind to IL-3 or IL-3Rα and inhibit signaling through IL-3, and bind to GM-CSF or GM-CSFα and inhibit signaling through GM-CSF. In this case, the monospecific compound binds to IL-5 or IL-5Rα and inhibits signaling through IL-5. Other combinations of compounds will be apparent to the skilled artisan.

In another embodiment, the present disclosure provides a method for preventing or treating ARDS in a subject comprising administering to the subject a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5 and GM-CSF.

The present disclosure also provides a compound that binds to CD131 and neutralizes signaling to a subject through IL-3, IL-5 and GM-CSF for use in treating or preventing ARDS in a subject. The present disclosure also provides the use of a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5 and GM-CSF in the manufacture of a medicament for treating or preventing ARDS in a subject.

According to the methods of the present disclosure, compounds that bind to CD131 and inhibit IL-3, IL-5 and GM-CSF signaling may be used to treat or prevent ARDS associated with any underlying condition. In some embodiments, the ARDS is associated with one or more of the following:

a) Infection;

b) Suction or aspiration of foreign matter (foreign substance);

c) A physical trauma; and

d) Inflammatory diseases.

In one embodiment, ARDS is associated with a viral infection. In one embodiment, ARDS is associated with a bacterial infection. In one embodiment, ARDS is associated with fungal infection. In one embodiment, ARDS is associated with sepsis.

In one embodiment, ARDS is associated with coronavirus infection.

In one embodiment, ARDS is associated with severe acute respiratory syndrome coronavirus (SARS-COV) infection. In one embodiment, the ARDS is associated with SARS-CoV-2 infection. Thus, in some embodiments, the subject has a coronavirus disease 2019 (covd-19). In particular, severe COVID-19 often results in ARDS. The methods of the present disclosure may be used to treat or prevent ARDS in severe covd-19 subjects. Thus, in some embodiments, the subject has severe COVID-19.

In some embodiments, ARDS is associated with aspiration or aspiration of the foreign body. For example, breathing high concentrations of smoke or chemical fumes can lead to ARDS, inhalation of vomit or near-drowning events (near-drowning) can also lead to ARDS.

In some embodiments, ARDS is associated with severe pneumonia. Severe cases of pneumonia often affect all five lobes of the lung and may lead to ARDS. Thus, in some embodiments, the subject has interstitial pneumonia.

In some embodiments, ARDS is associated with physical trauma. For example, head, chest and other major injuries may lead to ARDS. Accidents (such as falls or car accidents) can directly damage the lungs or the respiratory-controlling parts of the brain, leading to ARDS. In some embodiments, ARDS is associated with lung injury. In some embodiments, ARDS is associated with brain injury. In some embodiments, ARDS is associated with burns.

In some embodiments, ARDS is associated with surgery (e.g., cardiac surgery). For example, the subject being treated has undergone or will undergo a cardiac procedure, such as cardiopulmonary bypass.

In some embodiments, ARDS is associated with an inflammatory disease. For example, pancreatitis may lead to ARDS, as may other severe inflammatory diseases. In some embodiments, ARDS is associated with blood transfusion.

In some embodiments, the subject has ARDS.

In some embodiments, the subject meets Berlin definition of ARDS. Thus, in some embodiments, the subject has:

a) Onset of ARDS within 1 week or less of clinical injury or initial respiratory symptoms;

b) Acute hypoxemic respiratory failure, such as by ratio of arterial oxygen partial pressure to inhaled oxygen fraction (Pa 0) at Continuous Positive Airway Pressure (CPAP) or Positive End Expiratory Pressure (PEEP) of at least 5cm ₂ /FiO ₂ Ratio) of 300mmHg or less,

c) Bilateral shadows on chest radiographs, which cannot be explained entirely by effusion (fusion), solid changes, or lung tension; and

d) Respiratory failure, which cannot be explained entirely by heart failure or fluid overload (fluid overload).

In some embodiments of the methods of the present disclosure, the ARDS is mild ARDS. In some embodiments, the ARDS is a moderate ARDS. In some embodiments, the ARDS is a severe ARDS. The severity of ARDS can be categorized according to Berlin definition as follows:

(i) Mild ARDS: paO on at least 5cm CPAP or PEEP ₂ /FiO ₂ 200-300mmHg;

(ii) Moderate ARDS: paO on at least 5cm PEEP ₂ /FiO ₂ 100-200mmHg; and

(iii) Severe ARDS: paO on at least 5cm PEEP ₂ /FiO ₂ Less than or equal to 100mmHg.

Advantageously, the methods of the present disclosure may be used to prevent the onset of ARDS in addition to treating existing ARDS. Thus, in some embodiments, the subject does not have ARDS.

In some embodiments, the subject is at risk of developing ARDS. Methods of identifying a subject at risk of developing ARDS will be known to those of skill in the art and include those described herein.

In some embodiments, the subject has one or more or all of the following:

a) Respiratory rate of greater than 30 breaths per minute;

b) Oxygen saturation (SpO) of 93% or less in indoor air ₂ )；

c) Ratio of arterial oxygen partial pressure to inhaled oxygen fraction (PaO) of less than 300mmHg ₂ /FiO ₂ )；

d) SpO less than 218 ₂ /FiO ₂ Ratio of; and

e) Imaging lung infiltration in an amount greater than 50%.

In some embodiments, the above criteria may be used to assess whether a subject is at risk of developing ARDS.

In some embodiments, the subject does not receive High Flux Oxygen Therapy (HFOT) or non-invasive ventilation (NIV) at the time of administration of the compound that binds to CD 131.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce the severity of or prevent the onset of one or more symptoms of ARDS.

In one embodiment, the compound that binds to CD131 is administered in an amount sufficient to prevent death in or prior to an endotracheal tube. Endotracheal intubation is the process of inserting a tube (i.e., an endotracheal tube) through the mouth of a subject and into the airway so that the subject can be placed on a mechanical ventilator. Thus, the methods of the present disclosure also provide a method of preventing or reducing mechanical ventilation in a subject having ARDS, the method comprising administering to the subject a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5 and GM-CSF.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to effect one or more or all of the following:

a) Increase the number of days of survival and mechanical ventilation (free) time of the subject;

b) Shortening the hospitalization time (LOS) of the subject;

c) Improving the clinical status of the subject as assessed on the 8-point american society for allergy and infectious disease (National Institute ofAllergy and Infectious Disease) (NIAID) ordinal scale;

d) Reducing or preventing use of Continuous Positive Airway Pressure (CPAP) or bi-level positive airway pressure (BiPAP);

e) Reducing or preventing the use of High Flow Nasal Cannulas (HFNC);

f) Reducing or preventing the use of external membrane oxygenation (ECMO); and

g) Decreasing or preventing an increase in Sequential Organ Failure Assessment (SOFA) score for the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in inflammation in the lungs of the subject. In one embodiment, the compound that binds to CD131 is administered in an amount sufficient to enhance lung function.

In some embodiments, administration of a compound that binds to CD131 reduces or prevents an increase in one or more or all of the following:

a) The amount of neutrophils in the blood of the subject,

b) The amount of monocytes in the blood of the subject,

c) Neutrophil accumulation in the lungs of the subject,

d) Macrophage accumulation in the lungs of the subject,

e) Inflammation of the lungs of the subject,

f) Edema of the lungs of the subject,

g) NETosis of the lungs of the subject,

h) Myeloperoxidase activity in bronchoalveolar fluid (BALF) of the lungs of the subject,

i) The amount of protein in BALF of the lungs of the subject,

j) The amount of dsDNA in BALF of the lung of the subject, and

k) Lung injury score of the subject.

In some embodiments, administration of a compound that binds to CD131 reduces or prevents an increase in macrophage accumulation in the lungs of the subject. For example, macrophages are alveolar macrophages, monocyte-derived exuding macrophages and/or blood monocytes.

In some embodiments, administration of a compound that binds to CD131 reduces or prevents an increase in eosinophil accumulation in the lungs of the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in total cell count and/or total protein in BALF of the subject. In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in neutrophil levels present in BALF of the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in neutrophil elastase and/or myeloperoxidase activity levels in BALF of the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in the level of one or more or all of any of the following: G-CSF, plasminogen activator inhibitor-1 (PAI-1), D-dimer, neutrophil elastase, soluble AGE receptor (sRAGE), interferon gamma (IFN-gamma), interleukin 1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and tumor necrosis factor alpha (TNF-alpha). These biomarkers can be used to assess the efficacy of a treatment or to help identify subjects at risk of developing ARDS.

In some embodiments, the level of the protein is reduced or prevented from increasing in the lung of the subject. In some embodiments, the level of the protein is reduced or prevented from increasing in the blood of the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in the level of any or more or all of the following: C-C motif chemokine ligand 2 (CCL 2), CCL24, and IL-1 alpha.

In some embodiments, the level of the protein is reduced or prevented from increasing in the lung of the subject. In some embodiments, the level of the protein is reduced or prevented from increasing in the blood of the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in chemokine gene expression levels in the subject. For example, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in the expression level of the monocyte/macrophage chemoattractant factor Ccl2 gene and/or eosinophil chemokine Cc124 gene in the subject.

In some embodiments, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in the expression level of an inflammatory cytokine gene in the subject. For example, the compound that binds to CD131 is administered in an amount sufficient to reduce or prevent an increase in the expression level of the inflammatory cytokine Il1 gene in the subject.

In some embodiments, the expression level of the above-described genes is reduced or prevented from increasing in the lung of the subject. In some embodiments, the level of expression of the above-described gene is reduced or prevented from increasing in blood cells of the subject.

Furthermore, those of skill in the art will appreciate that the terms "reduce" and "prevent … … from increasing" refer herein to lower amounts of any of the items listed above relative to the amount in a subject prior to administration of a compound that binds to CD131 or relative to the amount in a corresponding control subject. For example, the control subject may be a subject receiving placebo and/or standard of care therapy instead of a compound that binds to CD 131.

In some embodiments, compounds that bind to CD131 and neutralize signaling through IL-3, IL-5, and GM-CSF prevent a decrease in the percentage of oxygenation of the subject's blood.

In some embodiments, a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF reduces the decrease in the percentage of blood oxygenation in a subject as compared to the decrease observed in a subject not administered the compound.

In one embodiment, the percent blood oxygenation of a subject administered a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF is at least about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15% greater than the percent blood oxygenation of a subject not administered a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF.

In one embodiment, the percent blood oxygenation of a subject administered a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF is about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%.

In one embodiment, the percent blood oxygenation of a subject administered a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF is about 90%.

In one embodiment, the percent blood oxygenation of a subject administered a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF is about 92%.

Methods for evaluating each of the foregoing are known in the art and/or described herein.

In some embodiments, the reduction in the amount of the items listed above, or the increased prevention thereof, or the reduction in the percentage of blood oxygenation, is assessed within 30 days of the first administration of the compound that binds to CD 131. In some embodiments, the reduction in the amount of the items listed above, or the prevention of an increase thereof, is assessed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 17, 21, 24, or 28 days after the first administration of the compound that binds to CD 131.

In one embodiment, compounds that bind to CD131 and neutralize signaling through IL-3, IL-5, and GM-CSF prevent an increase in blood Hemoglobin (HGB) levels in a subject.

In some embodiments, a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF reduces an increase in hemoglobin levels (HGB) in a subject as compared to an increase observed in a subject not administered the compound.

In one embodiment, the compound that binds to CD131 is at least 600nM or 500nM of IC ₅₀ Inhibit GM-CSF-induced proliferation of TF-1 cells. For example, IC ₅₀ At least about 400nM. For example, IC ₅₀ At least about 300nM or 200nM or 100nM. For example, IC ₅₀ At least about 50nM. For example, IC ₅₀ At least about 10nM or 5nM or 1nM. In one embodiment, an IC ₅₀ At least about 1nM. For example, IC ₅₀ At least about 0.9nM or 0.8nM or 0.6nM. In one embodiment, an IC ₅₀ At least about 0.5nM. In one embodiment, an IC ₅₀ At least about 0.4nM. In one embodiment, an IC ₅₀ At least about 0.3nM.

In one embodiment, the compound that binds to CD131 is at least 600nM or 500nM of IC ₅₀ Inhibit IL-5-induced proliferation of TF-1 cells. For example, IC ₅₀ At least about 400nM. For example, IC ₅₀ At least about 300nM or 200nM or 100nM. For example, IC ₅₀ At least about 50nM. For example, IC ₅₀ At least about 10nM or 5nM or 1nM. In one embodiment, an IC ₅₀ At least about 5nM. For example, IC ₅₀ At least about 4nM. In one embodiment, an IC ₅₀ At least about 4.5nM or at least about 4.6nM or at least about 4.7nM. In one embodimentIn (C), IC ₅₀ At least about 4.6nM.

In one embodiment, the compound that binds to CD131 is at least 600nM or 500nM of IC ₅₀ Inhibit IL-3-induced proliferation of TF-1 cells. For example, IC ₅₀ At least about 400nM. For example, IC ₅₀ At least about 300nM or 200nM or 100nM. For example, IC ₅₀ At least about 50nM. For example, IC ₅₀ At least about 10nM or 5nM or 1nM. In one embodiment, an IC ₅₀ At least about 1nM. For example, IC ₅₀ At least about 0.9nM or 0.8nM or 0.6nM. In one embodiment, an IC ₅₀ At least about 0.5nM. In one embodiment, an IC ₅₀ At least about 0.2nM or at least about 0.1nM. In one embodiment, an IC ₅₀ At least about 0.15nM.

Described herein are methods for determining an IC ₅₀ And comprising culturing TF-1 cells (e.g., about 1X 10) in the presence of a compound that binds to CD131 prior to the addition of the relevant growth factors (GM-CSF, IL-3, and/or IL-5) ⁴ TF-1 cells) (e.g., for at least about 3 minutes or 1 hour, such as about 30 minutes), and further culturing the cells (e.g., for at least about 48 hours or at least about 72 hours or at least about 96 hours, e.g., for about 72 hours), and then determining cell proliferation. Cell proliferation can be achieved by ³ [H]-growing the cells in the presence of thymidine for about 6 hours and determining ³ [H]Thymidine incorporation is determined, for example, by liquid scintillation counting. By determining proliferation of compounds that bind to CD131 at various concentrations, IC can be determined ₅₀ 。

In some embodiments, the compound that binds to CD 131:

a) Inhibiting GM-CSF-induced proliferation of TF-1 cells with an IC50 of at least 100 nM; and/or

b) Inhibiting IL-5-induced proliferation of TF-1 cells with an IC50 of at least 100 nM; and/or

c) IL-3-induced proliferation of TF-1 cells was inhibited with an IC50 of at least 100 nM.

In some embodiments, the compound that binds to CD 131:

a) Inhibiting GM-CSF-induced proliferation of TF-1 cells with an IC50 of at least 50 nM; and/or

b) Inhibiting IL-5-induced proliferation of TF-1 cells with an IC50 of at least 50 nM; and/or

c) IL-3-induced proliferation of TF-1 cells was inhibited with an IC50 of at least 50 nM.

In some embodiments, the compound that binds to CD 131:

a) Inhibiting GM-CSF-induced proliferation of TF-1 cells with an IC50 of at least 10 nM; and/or

b) Inhibiting IL-5-induced proliferation of TF-1 cells with an IC50 of at least 10 nM; and/or

c) IL-3-induced proliferation of TF-1 cells was inhibited with an IC50 of at least 10 nM.

In one embodiment, compounds that bind to CD131 reduce or prevent IL-3 and/or GM-CSF induced STAT-5 signaling.

In one embodiment, the compound that binds to CD131 is present in an IC of about 20nM or less ₅₀ Reduces or prevents IL-3 induced STAT-5 signaling. In one embodiment, pStat-5 IC ₅₀ IL-3 is about 10nM or less, or about 9nM or less, or about 8nM or less. In one embodiment, pStat-5 IC ₅₀ IL-3 is about 7.5nM or less, e.g., 7.3nM.

In one embodiment, the compound that binds to CD131 is present in an IC of about 60nM or less ₅₀ Reduces or prevents GM-CSF-induced STAT-5 signaling. In one embodiment, pStat-5 IC ₅₀ GM-CSF is about 50nM or less, or about 45nM or less, or about 40nM or less. In one embodiment, the compound that binds to CD131 is present in an IC of about 40nM ₅₀ Reduces or prevents GM-CSF-induced STAT-5 signaling.

For example, the compound may be contacted with a cell comprising a β -lactamase reporter gene (e.g., a TF-1 cell) under the control of an interferon regulatory factor 1 (irf 1) response element in the presence of IL-3 and/or GM-CSF. The cells are also contacted with a suitable substrate (e.g., a negatively charged fluorescent β -lactamase substrate, such as CCF2 or CCF 4) and the change in signal (e.g., fluorescence) is determined. A decrease in signal change in the positive control (i.e., cells contacted with IL-3 and/or GM-CSF in the absence of protein or antibody) indicates that the compound reduces or prevents IL-3 and/or GM-CSF-induced STAT-5 signaling.

In one embodiment, the compound that binds to CD131 has one or more of the following activities:

(i) Reducing or inhibiting activation of isolated human neutrophils by GM-CSF as determined by reducing or inhibiting GM-CSF-induced increase in neutrophil cell size;

(ii) Reducing or inhibiting IL-8 secretion induced by IL-3 of human basophils;

(iii) Reducing or preventing IL-3 mediated survival or plasmacytoid dendritic cells (pDCs);

(iv) Reducing or preventing activation of human peripheral blood eosinophils by IL-5, as determined by assessing changes in forward scatter assessed by flow cytometry;

(v) Reducing or preventing survival of human peripheral blood eosinophils in the presence of IL-5 and/or GM-CSF and/or IL-3;

(vi) Reducing or preventing IL-3 induced Tumor Necrosis Factor (TNF) alpha release from human mast cells;

(vii) Reducing or preventing IL-3 induced IL-13 release from human mast cells;

(viii) Reducing or preventing IgE-mediated enhancement of IL-8 release from human mast cells by IL-3 and/or IL-5 and/or GM-CSF;

(ix) Reducing or preventing the formation of colony forming units, granulocyte-macrophages (CFU-GM), by cd34+ human bone marrow cells cultured in the presence of Stem Cell Factor (SCF), GM-CSF, IL-3 and IL-5;

(x) Reducing the size or weight of polyps in a mouse xenograft model of human nasal polyposis; and/or

(xi) Reducing the number of B cells in polyps in a mouse xenograft model of human nasal polyposis.

In one embodiment, the compound that binds to CD131 does not significantly or significantly inhibit proliferation of TF-1 cells in response to one or more of erythropoietin, IL-6, IL-4, or stem cell factor. Described herein are methods for determining the ability of a compound that binds to CD131 to inhibit TF-1 cell proliferation relative to a cytokine or growth factor, and these methods are readily applicable to the present examples of the present disclosure.

In some embodiments, compounds useful in the present disclosure are proteins comprising an antigen binding site, e.g., a protein comprising an antigen binding site of an antibody or a single domain antibody. For example, the compound is an antibody or antigen binding domain thereof.

In some embodiments, the compound that binds to CD131 is a protein that comprises an antigen binding site that binds to CD 131. In some embodiments, the antigen binding site is an antigen binding site of an antibody or single domain antibody. In some embodiments, the antigen binding site comprises one or more CDRs.

Reference herein to a compound or protein or antibody that "binds to" CD131 provides literal support for a compound or protein or antibody that "specifically binds to (binds specifically to)" or "specifically binds to (specifically binds to)" CD 131.

In one embodiment, when the polypeptide is immobilized on a solid surface and K _D When determined by surface plasmon resonance, the nucleic acid sequence comprising SEQ ID NO:5, K of a protein of a polypeptide of the sequence shown in FIG. 5 _D About 10nM or less.

In one embodiment, K _D Is 10nM or less, e.g., 5nM or less, or 4nM or less, or 3nM or less, or 2nM or less. In one embodiment, K _D 1nM or less. In one embodiment, K _D 0.9nM or less, or 0.7nM or less, or 0.8nM or less, or 0.7nM or less, or 0.6nM or less. In one embodiment, K _D 0.5nM or less. In one embodiment, K _D 0.4nM or less. In one embodiment, K _D 0.3nM or less. In one embodiment, K _D 0.2nM or less.

In one embodiment, the protein comprising the antigen binding site is at a K of about 10nM or less _D Binding to cells expressing CD131 (e.g., neutrophils Granulocytes or eosinophils or TF-1 cells), for example, using competition assays with labeled and unlabeled proteins or antibodies. In one embodiment, K _D 5nM or less, or 4nM or less, or 3nM or less, or 2nM or less. In one embodiment, K _D 1nM or less. In one embodiment, K _D 0.9nM or less, or 0.7nM or less, or 0.8nM or less, or 0.7nM or less, or 0.6nM or less.

In one embodiment, for neutrophils, K _D About 300nM or less.

In one embodiment, for eosinophils, K _D About 700nM or less.

In one embodiment, for TF-1 cells, K _D About 400nM or less.

In one embodiment, the protein comprising an antigen binding site is a protein comprising one or more antibody variable regions. In one embodiment, the protein comprises a heavy chain variable region (V _H ). In one embodiment, the protein comprises a light chain variable region (V _L ). In one embodiment, the protein comprises V _H And V _L . In some embodiments, V _H And V _L In the same polypeptide chain. In other embodiments, V _H And V _L In separate polypeptide chains.

In some embodiments, the protein is a single domain antibody (sdAb).

In some embodiments, the protein comprises an Fv.

In some embodiments, the protein comprises:

(i) Single chain Fv fragments (scFv);

(ii) Dimeric scFv (di-scFv); or (b)

(iii) A double body;

(iv) A trisome;

(v) A tetrahedron;

(vi)Fab；

(vii)F(ab′) ₂ ；

(viii)Fv；

(ix) And resistConstant region of body, fc or heavy chain constant domain (C _H ) 2 and/or C _H 3 to one of (i) to (viii);

(x) One of (i) to (viii) linked to albumin or a functional fragment or variant thereof or a protein that binds to albumin; or (b)

(xi) An antibody.

In some embodiments, the protein is selected from the group consisting of:

(i) Single chain Fv fragments (scFv);

(ii) Dimeric scFv (di-scFv); or (b)

(iii) A double body;

(iv) A trisome;

(v) A tetrahedron;

(vi)Fab；

(vii)F(ab′) ₂ ；

(viii)Fv；

(ix) With the constant region, fc or heavy chain constant domain (C) _H ) 2 and/or C _H 3 to one of (i) to (viii);

(x) One of (i) to (viii) linked to albumin, a functional fragment or variant thereof, or a protein that binds to albumin (e.g., an antibody or antigen binding fragment thereof); or (b)

(xi) An antibody.

In one embodiment, the protein comprises an Fc region.

In one embodiment, the protein comprises one or more amino acid substitutions that increase the half-life of the protein. In one embodiment, an antibody comprises an Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region for a neonatal Fc receptor (FcRn).

In one embodiment, the protein is an antibody, such as a monoclonal antibody. In one embodiment, the antibody is a naked antibody.

In one embodiment, the protein (or antibody) is chimeric, deimmunized, humanized, human or primatized.

In one embodiment, the protein or antibody is human.

Exemplary antibodies include 9A2-VR24.29 (also referred to as "CSL 311") described in WO 2017/088028 and Blood (Blood) 94 in Sun et al (1999): 1943-1951, BION-1.

In one embodiment, the protein comprises a human constant region, for example an IgG constant region, such as an IgG1, igG2, igG3 or IgG4 constant region, or a mixture thereof. In the case of proteins comprising V _H And V _L In the case of V _H May be linked to the heavy chain constant region, and V _L May be linked to the light chain constant region.

For example, during production or purification of a protein or antibody, or by recombinant engineering of a nucleic acid encoding a heavy chain, whole antibodies (or comprising constant regions or C's) may be removed _H 3) a heavy chain constant region of a heavy chain. Thus, a whole antibody (or CD131 binding compound) may comprise a population with all C-terminal lysine residues removed, a population with no C-terminal lysine residues removed, and/or a population with a mixture of proteins with and without C-terminal lysine residues. In some embodiments, the population may additionally comprise proteins in which a C-terminal lysine residue is removed in one of the heavy chain constant regions. Similarly, a composition of whole antibodies may comprise a mixture of identical or similar populations of antibodies with or without C-terminal lysine residues.

In one embodiment, the protein is in a composition. For example, the composition comprises a protein comprising an antigen binding site or an antibody as described herein. In one embodiment, the composition additionally comprises one or more variants of the protein or antibody. For example, it comprises variants lacking the encoded C-terminal lysine residue, deamidated variants and/or glycosylated variants and/or variants comprising pyroglutamic acid (pyroglutamic acid), e.g. at the N-terminus of the protein, and/or variants lacking the N-terminal residue, e.g. N-terminal glutamine in the antibody or V-region, and/or variants comprising all or part of the secretion signal. Deamidated variants of the encoded asparagine residue may lead to the production of iso-aspartic acid and aspartic acid isoforms, or even succinamides involving adjacent amino acid residues. Deamidated variants of the encoded glutamine residues can produce glutamate. When referring to a particular amino acid sequence, compositions comprising heterogeneous mixtures of such sequences and variants are also intended to be included.

In one embodiment, a protein or antibody as described herein comprises the constant region of an IgG4 antibody or the stabilized constant region of an IgG4 antibody. In one embodiment, the protein or antibody comprises an IgG4 constant region having a proline at position 241 (according to the numbering system of Kabat et al (sequence of protein of immunological interest (Sequences ofProteins ofImmunological Interest), washington, inc. (United States Department of Health and Human Services), 1987 and/or 1991)).

In one embodiment, the heavy chain constant region comprises SEQ ID NO: 16. In one embodiment, the protein or protein-containing composition comprises a heavy chain constant region, including a stabilized heavy chain constant region, comprising a mixture of sequences with or without a C-terminal lysine residue, either in whole or in part.

In some embodiments, the protein comprises an antibody variable region that binds to CD131 and competitively inhibits binding of antibody 9A2-VR24.29 to CD131, said antibody 9A2-VR24.29 comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:6, V of the sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO:7, V of the sequence shown in FIG. 7 _L 。

In some embodiments, the protein comprises an antibody variable region that binds to CD131 and competitively inhibits binding of antibody 9A2-VR24.29 to CD131, said antibody 9A2-VR24.29 comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:6, V of the sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO:18, V of the sequence shown in SEQ ID NO. 18 _L 。

In some embodiments, the protein binds to the same or overlapping epitope in CD131 as antibody 9A2-VR24.29 against CD131, which antibody 9A2-VR24.29 comprises a polypeptide comprising SEQ ID NO:6, V of the sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO: 7V of the sequence shown in FIG. 7 _L 。

In some embodiments, the protein binds to the same or overlapping table in CD131 as antibody 9A2-VR24.29 against CD131 At position, the antibody 9A2-VR24.29 comprises a heavy chain comprising SEQ ID NO:6, V of the sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO:18, V of the sequence shown in SEQ ID NO. 18 _L 。

In some embodiments, the antigen binding site binds to an epitope within site 2 of CD 131. In this regard, the skilled artisan will recognize that site 2 of CD131 is comprised of residues from two CD131 polypeptides that form a dimer, e.g., site 2 comprises residues within loops A-B and E-F of domain 1 of one CD131 polypeptide and residues within loops B-C and F-G of the other CD131 polypeptide.

In some embodiments, the antigen binding site binds to an epitope formed upon dimerization of two CD131 polypeptides.

In some embodiments, the antigen binding site binds to a residue within domain 1 of a CD131 polypeptide and a residue within domain 4 of another CD131 polypeptide. In one embodiment, the residue within domain 1 of CD131 comprises SEQ ID NO:1, and/or residues within domain 4 of CD131 comprise the amino acid sequence of SEQ ID NO: residues within the region 364-367 of 1.

In some embodiments, the protein binds to an epitope comprising:

a) Corresponding to SEQ ID NO:1, amino acids in one CD131 polypeptide chain of one or more or all of positions 39, 101, 102, 104, 105, 106 and 107, and

b) Corresponding to SEQ ID NO:1, amino acids in one or more or all of positions 364, 365, 366, 367, 420 and 421 of another CD131 polypeptide chain.

In some embodiments, the protein comprises an antibody variable region comprising V _H And V _L The V is _H Comprising a polypeptide comprising SEQ ID NO:6, V of the amino acid sequence shown in FIG. 6 _H Is set forth in (2), the V _L Comprising a polypeptide comprising SEQ ID NO:7, V of the amino acid sequence shown in FIG. 7 _L Is a CDR of (c).

In some embodiments, the protein comprises an antibody variable region comprising V _H And V _L The V is _H Comprises the following componentsComprising SEQ ID NO:6, V of the amino acid sequence shown in FIG. 6 _H Is set forth in (2), the V _L Comprising a polypeptide comprising SEQ ID NO:18, V of the amino acid sequence shown in SEQ ID NO. 18 _L Is a CDR of (c).

In some embodiments, the protein comprises:

a)V _H the V is _H Comprising HCDR1, said HCDR1 comprising or consisting of SEQ ID NO:8 or the amino acid sequence set forth in SEQ ID NO:8 having a sequence composition of no more than one or two or three amino acid substitutions; HCDR2, said HCDR2 comprising or consisting of SEQ ID NO:9 or the amino acid sequence shown relative to SEQ ID NO:9 having a sequence composition of no more than one or two or three amino acid substitutions; and HCDR3, said HCDR3 comprising or consisting of SEQ ID NO:10 or the amino acid sequence set forth in SEQ ID NO:10 having a sequence composition of no more than one or two or three amino acid substitutions; and

b)V _L The V is _L Comprising an LCDR1, said LCDR1 comprising or consisting of SEQ ID NO:11 or a sequence having NO more than one or two or three amino acid substitutions relative to SEQ ID NO: 11; LCDR2, said LCDR2 comprising or consisting of SEQ ID NO:12 or the amino acid sequence shown relative to SEQ ID NO:12 having a sequence composition of no more than one or two or three amino acid substitutions; and LCDR3, said LCDR3 comprising or consisting of SEQ ID NO:13 or the amino acid sequence set forth relative to SEQ ID NO:13 has a sequence composition of no more than one or two or three amino acid substitutions.

In some embodiments, the protein comprises:

a)V _H the V is _H Comprising HCDR1, said HCDR1 comprising or consisting of SEQ ID NO:8, and a polypeptide comprising the amino acid sequence shown in seq id no; HCDR2, said HCDR2 comprising or consisting of SEQ ID NO:9, and a polypeptide comprising the amino acid sequence shown in seq id no; HCDR3, said HCDR3 comprising or consisting of SEQ ID NO:10, and a polypeptide comprising the amino acid sequence shown in seq id no; and

b)V _L the V is _L Comprising an LCDR1, said LCDR1 comprising or consisting of SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no; LCDR2, said LCDR2 comprising or consisting of SEQ ID NO: shown in 12Is composed of amino acid sequences of (2); and LCDR3, said LCDR3 comprising or consisting of SEQ ID NO:13, and a polypeptide comprising the amino acid sequence shown in seq id no.

In some embodiments, the protein comprises V _H And V _L The V is _H Comprising a sequence identical to SEQ ID NO:6, said V has an amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity to said V _L Comprising a sequence identical to SEQ ID NO:7 has an amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity.

In some embodiments, the protein comprises V _H And V _L The V is _H Comprising a sequence identical to SEQ ID NO:6, said V has an amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity to said V _L Comprising a sequence identical to SEQ ID NO:18 has an amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity.

In some embodiments, the protein comprises a polypeptide comprising SEQ ID NO:6, V of the amino acid sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO:7, V of the amino acid sequence shown in FIG. 7 _L 。

In some embodiments, the protein comprises a polypeptide comprising SEQ ID NO:6, V of the amino acid sequence shown in FIG. 6 _H And a polypeptide comprising SEQ ID NO:18, V of the amino acid sequence shown in SEQ ID NO. 18 _L 。

In some embodiments, the protein comprises a heavy chain comprising a sequence identical to SEQ ID NO:14, and the light chain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO:15, an amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity.

In some embodiments, the protein comprises a polypeptide comprising SEQ ID NO:14 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain of the amino acid sequence shown in seq id no.

In one embodiment, the compound that binds to CD131 is administered in combination with another therapy.

In one embodiment, another therapy includes administration of an anti-inflammatory compound. In one embodiment, another therapy includes administration of an immunomodulatory or immunosuppressive agent.

In some embodiments, another therapy comprises administering a protein comprising an antigen binding site. In some embodiments, the protein comprising an antigen binding site is an antibody.

In some embodiments, the compound that binds to CD131 is administered in combination with a cell. In some embodiments, the cell is a stem cell, such as a mesenchymal stem cell.

In some embodiments, the compound that binds to CD131 is administered in combination with gene therapy.

In one embodiment, the compound that binds to CD131 is administered concurrently with another therapy. In one embodiment, the compound that binds to CD131 is administered prior to another therapy. In one embodiment, the compound that binds to CD131 is administered after another therapy.

In some embodiments, the compound that binds to CD131 is administered in combination with standard of care therapy. The standard of care regimen may be the standard of care regimen of the underlying etiology of ARDS, or it may be the standard of care regimen of ARDS itself.

In some examples, the standard of care regimen includes one or more or all of the following:

a) A prone position;

b) Fluid management;

c) Administering nitric oxide;

d) Administering a neuromuscular blocking agent;

e) Artificial ventilation;

f) Oxygenation of the outer membrane; and

g) An antiviral agent or antibiotic is administered.

In one embodiment, standard of care therapy includes administration of an antiviral drug. In one embodiment, standard of care therapy comprises administration of adefovir (remdesivir). In one embodiment, standard of care therapy includes administration of one or more of the following:

a) Hydroxychloroquine;

b) Chloroquine;

c) Lopinavir (lopinavir);

d) Ritonavir (ritonavir);

e) Azithromycin;

f) Interferon beta;

g) Anakinra (anakinra);

h) Tobalizumab (tocilizumab);

i) Sha Lilu mab (sarilumab);

j) Dexamethasone (dexamethasone);

k) Aspirin;

l) losartan;

m) simvastatin (simvastatin); and

n) baratinib (baricitinib).

In one embodiment, the subject is a human. In one embodiment, the subject is an adult, e.g., over 18 years old. In one embodiment, the subject is a child, e.g., less than 18 years old. In one embodiment, the subject is between 18 and 90 years old. In one embodiment, the subject is between 50 and 80 years old.

In one embodiment, the subject does not have Chronic Obstructive Pulmonary Disease (COPD). In one embodiment, the subject does not have asthma.

Keywords of sequence Listing

SEQ ID NO 1: amino acid sequence of homo sapiens CD131

SEQ ID NO 2: amino acid sequence of homo sapiens IL-3 receptor alpha

SEQ ID NO 3: amino acid sequence of human homo sapiens GM-CSF receptor

SEQ ID NO 4: amino acid sequence of the homo sapiens IL-5 receptor

SEQ ID NO 5: amino acid sequence of soluble homo sapiens CD131 comprising a C-terminal 6xHis tag

SEQ ID NO 6: v of 9A2-VR24.29 _H Amino acid sequence of (2)

SEQ ID NO 7: v of 9A2-VR24.29 _L Amino acid sequence of (2)

SEQ ID NO 8: amino acid sequence of HCDR1 of 9A2-VR24.29

SEQ ID NO 9: amino acid sequence of HCDR2 of 9A2-VR24.29

SEQ ID NO 10: amino acid sequence of HCDR3 of 9A2-VR24.29

SEQ ID NO 11: amino acid sequence of LCDR1 of 9A2-VR24.29

SEQ ID NO 12: amino acid sequence of LCDR2 of 9A2-VR24.29

SEQ ID NO 13: amino acid sequence of LCDR3 of 9A2-VR24.29

SEQ ID NO 14: amino acid sequence of heavy chain of 9A2-VR24.29

The amino acid sequence of the light chain of SEQ ID NO 15:9A 2-VR24.29

SEQ ID NO 16: amino acid sequence of stabilized IgG4 heavy chain constant region

SEQ ID NO 17: amino acid sequence of K light chain constant region

SEQ ID NO 18: v of 9A2-VR24.29 _L Amino acid sequence of (2)

Drawings

FIG. 1 is a graphical representation of a series of stacked columns of (A) cell numbers and (B) differential cells per milliliter (mean +/-SEM) of lymphocytes, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) of mice prophylactically administered chimeric monoclonal antibodies. Mice were intravenously administered 50mg/kg of chimeric monoclonal antibody (chimeric mAb) or isotype control (BM 4) 24 hours before cannulation with 3 μg LPS or PBS as described in example 1. BALF of mice was collected 24 hours after intubation for analysis. Significant differences in cell numbers between chimeric mAb and BM4 groups were observed (common one-way anovachunett multiple comparison test) (p=0.0004); n=3 (PBS), 6 (bm4_lps) and 6 (chimeric mab_lps).

Figure 2 is a graphical representation of a series of stacked columns of (a) cell numbers and (B) differential cells per milliliter (mean +/-SEM) of lymphocytes, neutrophils, and macrophages in bronchoalveolar lavage (BALF) of mice therapeutically administered with the chimeric monoclonal antibodies described in example 1. At 6 hours after cannulation with 3 μg of LPS or PBS, mice were intravenously administered 50mg/kg of chimeric monoclonal antibody (chimeric mAb) or isotype control (BM 4). BALF of mice was collected 24 hours after intubation for analysis. Significant differences in cell numbers between chimeric mAb and BM4 groups were observed (common one-way anovachunett multiplex comparison test) (p=0.0013); n=3 (PBS), 5 (bm4_lps) and 6 (chimeric mab_lps).

FIG. 3 is a series of graphical representations showing the inhibition of (A) GM-CSF and (B) IL-3 induced FDCP1 cell proliferation by a chimeric monoclonal antibody (chimeric mAb) as compared to an isotype control monoclonal antibody (isotype control mAb) described in example 2.

Fig. 4 is a series of graphical representations showing markers of lung leukocyte count and lung injury in the ARDS mouse model described in example 3. The figure: (a) percent weight loss of hβ cTg mice administered with LPS-ISO or CSL311, (B) blood granulocyte count of mice administered with LPS-ISO or CSL311, (C) blood monocyte count, (D) bronchoalveolar lavage (BALF) neutrophil count. (E) Immunohistochemical staining (H & E) sections of lungs from H beta cTg mice administered with LPS-ISO or CSL 311. The graphical representation shows (F) lung injury scores based on H & E staining of lung sections, (G) lung MPO activity and (H) BALF protein in mice administered with LPS-ISO or CSL 311. N=6-7, < 0.05, anova versus LPS-ISO group.

FIG. 5 is a series of graphical representations showing netosis in a mouse model of ARDS described in example 3. (A) Immunohistochemical staining of lung sections of h.beta. cTg mice administered with LPS-ISO or CSL 311. MPO (green) and histone H3 (red) determine the region of netosis based on co-localization of MPO and histone H3. Graphical representation of (B) MPO activity (C) dsDNA (D) MPO activity (E) dsDNA in BALF of hβ cTg mice administered LPS-ISO or CSL 311. N=6-7, < 0.05, anova versus LPS-ISO group.

Fig. 6 is a graph showing the percent blood oxygenation in a mouse model of ARDS described in example 3. Blood oxygenation was determined in mice administered with h.beta. cTg of PBS (and no LPS), LPS-ISO or LPS-CSL 311. Data are expressed as oxygen saturation% +/-SEM and statistical analysis of the LPS-ISO and LPS-CSL311 groups were compared using two-way ANOVA and Bonferroni multiple post-comparison tests. * Represents p < 0.001; * Represents p < 0.01; * Representing p < 0.05. N=5 mice per group.

Fig. 7 is a series of graphical representations showing that βc cytokines and βc receptors are overexpressed in IAV-infected hβ cTg mice. Mice were infected with h.beta. cTg with IAV (104 PFU, HKx31 strain) and slaughtered on day 3 and day 6 post infection. Beta C cytokine- (A) Gm-csf, (B) Il3, (C) Il5; and gene expression of βc receptor- (D) CSF2RB was analyzed by RTqPCR in lung tissue. n=6; data are mean ± SE; * p < 0.05, p < 0.01, p < 0.001 single-factor ANOVA.

Fig. 8 is a series of graphical representations showing that CSL311 reduced pulmonary inflammation and injury without compromising viral clearance in IAV-infected hβ cTg mice. On day 4 post infection, CSL311 or isotype control (ISO) was administered to IAV infected hβ cTg mice. On day 7 IAV induced (a) weight loss, which was not significantly improved by CSL311 treatment. (B) Pulmonary viral load was measured by RTqPCR against viral PA gene and no difference was detected between IAV-infected CSL311 and ISO-treated mice. Elevated levels of (C) blood mononuclear cells, (D) blood neutrophils, and (E) blood Hemoglobin (HGB) induced by IAV infection were significantly reduced by CSL 311. (F) BAL neutrophils and (G) BAL macrophages were also reduced in the case of CSL311 treatment. n=6; data are mean ± SE, except viral load is median ± quartile range; * p < 0.05, < p < 0.01, < p < 0.001 single-factor ANOVA, except for viral load as measured by Mann-Whitney U.

Fig. 9 is a series of graphical representations showing that CSL311 blocks lung myeloid cells and protects NK cells and T cells in IAV-infected hβ cTg mice. Flow cytometry analysis using the gating strategy of (a) myeloid cells showed that lung (B) neutrophils, (C) Alveolar Macrophages (AM), (D) Exudative Macrophages (EM), (E) monocytes and (F) eosinophils were significantly increased in hβ cTg mice at day 6 post IAV infection, and significantly decreased in the case of CSL311 treatment. Flow cytometry analysis using (G) gating strategies for profiling lymphoid cells showed that lung (H) NK cells, (I) NKT cells and (J) regulatory T cells (tregs) increased after IAV infection and were not affected by CSL311 treatment. In the lung there was no difference in (K) lung CD4 cell numbers and (L) CD8 cell numbers in all groups; n=6; data are mean ± SE; * p < 0.05, p < 0.01, p < 0.001 single-factor ANOVA.

Fig. 10 is a series of graphical representations showing that CSL311 attenuated pulmonary cytokine storm and maintained interferon expression in IAV infected hβ cTg mice. The expression of neutrophil chemokine (a) Cxcl1, monocyte/macrophage chemokine (B) Ccl2, eosinophil chemokine (C) Ccl24, T cell/NK cell chemokine (D) Cxcl10 in the lung of IAV infected mice was detected by RTqPCR, wherein CSL311 significantly reduced the Ccl2 and Ccl24 levels. In IAV infected mice, the expression of the pro-inflammatory genes (E) Il1a and (F) Il6 was also increased, and CSL311 treatment significantly reduced the expression of Il1a, but not Il 6. (G) Type I interferon (Ifnb), (H) type II interferon (Ifng) and type III interferon (Ifnl 2/3) were also significantly induced in the lung of IAV-infected mice in a manner that was not significantly altered by CSL311 treatment; n=6; * p is less than 0.05, and the data are average value +/-SE; * P < 0.01, p < 0.001 single factor ANOVA.

Detailed Description

General description

Throughout this specification, unless the context requires otherwise, reference to a single step, a combination of materials, a group of steps or a group of combinations of materials is to be understood as covering one or more (i.e. one or more) of such steps, combinations of materials, groups of steps or groups of combinations of materials.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The scope of the present disclosure is not limited by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure.

Any embodiment of the disclosure herein should be considered as applicable to any other embodiment of the disclosure mutatis mutandis unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, recombinant proteins, cell culture and immunological techniques used in the present disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J.Perbal, molecular cloning Utility Specification (A Practical Guide to Molecular Cloning), john Willi parent-child company (John Wiley and Sons) (1984), J.Sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbour Laboratory Press) (1989), T.A.Brown (eds.), "basic molecular biology: practical methods (Essential Molecular Biology: A Practical Approach), volumes 1 and 2, IRL Press (1991), D.M.Glover and B.D.Hames (editors), "DNA cloning: methods of use (DNA Cloning: A Practical Approach), volumes 1-4, IRL Press (1995 and 1996), and F.M.Ausubel et al (editions), "Current protocols for molecular biology (Current Protocols in Molecular Biology)," Greene Pub.associates and Wiley-Interscience (1988, including all updates to date), ed Harlow and David Lane (editions), "antibodies: laboratory Manual (Antibodies: A Laboratory Manual), cold spring harbor laboratory (1988), and J.E. Coligan et al (edit) Current immunology protocol (Current Protocols in Immunology), john Willi parent, including all updates to date.

The variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof may be described and defined herein by the Kabat sequence (Kabat Sequences of Proteins of Immunological Interest) of proteins of immunological interest, national institutes of health (National Institutes of Health), besseda (Bethesda, md.), 1987 and 1991, bork et al, journal of molecular biology (J mol. Biol.) 242, 309-320, 1994, chothia and Lesk journal of molecular biology 196: the discussion in 901-917, 1987, chothia et al, nature 342, 877-883, 1989 and/or A1-Lazikani et al, journal of molecular biology 273, 927-948, 1997 is further elucidated.

The term "and/or", e.g. "X and/or Y", shall be understood to mean "X and Y" or "X or Y", and shall be used to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected definition

For naming purposes only and not for limitation, exemplary sequences of human CD131 (pre-CD 131) are set forth in NCBI reference sequences: NP-000386.1 and NCBI Genbank accession number P32927 (and shown in SEQ ID NO: 1). Mature human CD131 lacks the sequence of SEQ ID NO:1 and amino acids 1-16. The position of the amino acid is generally referred to herein by reference to pre-CD 131. The position in mature CD131 is readily determined by consideration of the signal sequence (amino acids 1-16 in the case of SEQ ID NO: 1). The sequences of CD131 from other species may be determined using the sequences provided herein and/or in publicly available databases and/or using standard techniques (e.g., as described in Ausubel et al (eds.), greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (1989). Mention of human CD131 may be abbreviated as hCD131. Reference to soluble CD131 refers to a polypeptide comprising the extracellular region of CD131, e.g., SEQ ID NO:1 from amino acids 17 to 438.

Reference herein to CD131 includes natural forms of CD131 as well as mutant forms thereof that retain the ability to bind to CD131 (e.g., hCD 131) and induce signaling. CD131 is also known as "CSF2RB" and "cytokine receptor co-subunits β" and "β (β) co-receptors" (abbreviated as "βcr" or "βc").

As contemplated by the present disclosure, a "compound" may take any of a variety of forms, including natural compounds, chemical small molecule compounds, or biological compounds, or macromolecules. Exemplary compounds include antibodies or proteins, nucleic acids, polypeptides, peptides and small molecules comprising antigen binding fragments of antibodies.

As used herein, the term "disease" or "condition" refers to the disruption or interference of normal function and is not limited to any particular condition, disease, or disorder.

As used herein, the terms "treatment", "treatment" or "treatment" include administration of a compound described herein to reduce, prevent or eliminate at least one symptom of a particular disease or condition.

As used herein, the term "preventing", "prevention" or "prevention" includes administration of a compound described herein, thereby preventing or impeding the development of at least one symptom of a condition, e.g., before the symptom has fully developed in a subject.

As used herein, the term "subject" should be understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. In one embodiment, the subject is a human.

The term "protein" should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., polypeptide complexes) covalently or non-covalently linked to each other. For example, a series of polypeptide chains may be covalently linked using suitable chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. In some embodiments, the protein is a fusion protein. As used herein, a "fusion protein" is a protein comprising at least two domains that have been linked such that they translate as a single unit, producing a single protein.

According to the preceding paragraphs, the term "polypeptide" or "polypeptide chain" is understood to mean a series of consecutive amino acids linked by peptide bonds.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that is not associated with a naturally associated component with which it is associated in its natural state due to its origin or derived source; substantially free of other proteins from the same source. Proteins may be substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. By "substantially purified" is meant that the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

The term "recombinant" is understood to mean the product of artificial gene recombination. Thus, in the context of recombinant proteins comprising an antibody antigen binding domain, this term does not encompass naturally occurring antibodies in vivo in a subject, which are the products of natural recombination that occur during B cell maturation. However, if such an antibody is isolated, it is considered to be an isolated protein comprising an antibody antigen binding domain. Similarly, if recombinant means are used to isolate and express nucleic acids encoding the protein, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. Recombinant proteins also encompass proteins that are expressed by artificial recombinant means when they are in a cell, tissue or subject (e.g., when it is expressed therein).

As used herein, the term "antigen binding site" should be understood to mean a structure formed by a protein capable of binding or specifically binding to an antigen. The antigen binding site need not be a series of consecutive amino acids, or even amino acids in a single polypeptide chain. For example, in Fv produced from two different polypeptide chains, the antigen bindsThe binding site is defined by V _L And V _H Are formed of a series of amino acids that interact with the antigen and are typically, but not always, located in one or more CDRs in each variable region. In some embodiments, the antigen binding site is or comprises V _H Or V _L Or Fv. In some embodiments, the antigen binding site comprises one or more CDRs of an antibody.

Those skilled in the art will appreciate that an "antibody" is generally considered to be comprised of multiple polypeptide chains (e.g., comprising V _L Polypeptide of (c) and comprising V _H Polypeptide of (c) can be constituted by variable region proteins. Antibodies typically also comprise constant domains, some of which may be arranged into constant regions, in the case of heavy chains, including constant or crystallizable fragments (fcs). V (V) _H And V _L To form Fv comprising an antigen binding region capable of specifically binding to one or more closely related antigens. Typically, the light chain from a mammal is a kappa light chain or a lambda light chain, and the heavy chain from a mammal is alpha, delta, epsilon, gamma, or mu. Antibodies can be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG) ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ ) Or subclasses. The term "antibody" also encompasses humanized antibodies, primatized antibodies, human antibodies and chimeric antibodies.

The terms "full length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in substantially its intact form relative to an antigen-binding fragment of the antibody. In particular, whole antibodies include those having heavy and light chains including an Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

As used herein, "variable region" refers to the portion of the light and/or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen and includes Complementarity Determining Regions (CDRs), namely CDR1, CDR2 and CDR3, and Framework Regions (FR)Amino acid sequence. Exemplary variable regions comprise three or four FR (e.g., FRi, FR2, FR3, and optionally FR 4) and three CDRs. In the case of proteins derived from IgNAR, the protein may lack CDR2.V (V) _H Refers to the variable region of the heavy chain. V (V) _L Refers to the variable region of the light chain.

As used herein, the term "complementarity determining region" (synonymous CDRs; i.e., CDR1, CDR2, and CDR 3) refers to the amino acid residues of an antibody variable region, the presence of which is necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR 3. Amino acid positions assigned to CDRs and FRs may be defined according to the "Kabat sequence of proteins of immunological interest", national institutes of health, bezidas, md., 1987 and 1991, or other numbering systems in the practice of the present disclosure, e.g., chothia and Lesk, J.Molec.Biol., 196:901-917, 1987Chothia et al, nature 342, 877-883, 1989, below; and/or Al-Lazikani et Al, journal of molecular biology, 273:927-948, 1997; lefranc et al, "development and comparative immunology (development. And company. Immunol.), 27:55-77, 2003; or Honnegher and Plukthhun journal of molecular biology, 309:657-670, 2001. For example, according to the numbering system of Kabat, V _H The Framework Regions (FR) and CDRs are located as follows: residues 1-30 (FR 1), 31-35 (CDR 1), 36-49 (FR 2), 50-65 (CDR 2), 66-94 (FR 3), 95-102 (CDR 3) and 103-113 (FR 4). According to the numbering system of Kabat, V _L FR and CDR are located as follows: residues 1-23 (FRI), 24-34 (CDR 1), 35-49 (FR 2), 50-56 (CDR 2), 57-88 (FR 3), 89-97 (CDR 3) and 98-107 (FR 4). The present disclosure is not limited to FR and CDR as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one embodiment, references herein to CDRs (or FR) are with respect to those regions according to the Kabat numbering system.

"framework region" (FR) is a variable region residue other than a CDR residue.

As used herein, the term "Fv" is understood to mean any protein, whether composed of multiple polypeptides orConsists of a single polypeptide, wherein V _L And V _H Associates and forms a complex with an antigen binding site, i.e. is capable of specifically binding to an antigen. V forming an antigen binding site _H And V _L May be in a single polypeptide chain or in different polypeptide chains. In addition, fv of the present disclosure (as well as any protein of the present disclosure) may have multiple antigen binding sites that may or may not bind to the same antigen. This term should be understood to encompass fragments derived directly from antibodies as well as proteins corresponding to such fragments produced using recombinant means. In some embodiments, V _H Is not associated with the heavy chain constant domain (C _H ) 1 connection, and/or V _L Is not associated with the constant domain of the light chain (C _L ) And (5) connection. Exemplary Fv-containing polypeptides or proteins include Fab fragments, fab 'fragments, F (ab') fragments, scFv, diabodies, triabodies, tetrads or higher complexes, or linked to a constant region or domain thereof (e.g., C _H 2 or C _H 3 domain), for example minibodies. "Fab fragments" consist of monovalent antigen binding fragments of immunoglobulins and can be produced by digestion of whole antibodies with papain to produce fragments consisting of the complete light chain and a portion of the heavy chain, or can be produced using recombinant means. The "Fab' fragment" of an antibody can be produced by treating the whole antibody with pepsin, followed by reduction to produce a polypeptide consisting of the complete light chain and containing V _H And a portion of the heavy chain of a single constant domain. Two Fab' fragments were obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. The "F (ab ') 2 fragment" of an antibody consists of a dimer of two Fab' fragments bound together by two disulfide bonds, and is obtained by treating the whole antibody molecule with pepsin without subsequent reduction. "Fab ₂ A "fragment" is a recombinant fragment comprising two Fab fragments which are fused together using, for example, a leucine zipper or C _H 3 domain linkage. A "single chain Fv" or "scFv" is a recombinant molecule comprising an antibody variable region fragment (Fv) in which the light and heavy chain variable regions are joined by a suitable flexibilityPolypeptide linkers are covalently linked.

As used herein, the term "bind" with respect to the interaction of a compound or antigen binding site thereof with an antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures, rather than to general proteins. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" that binds to the antibody in a reaction containing labeled "A" and protein.

As used herein, the term "specifically bind (specifically binds)" or "specifically bind (binds specifically)" should be understood to mean that the compound of the present disclosure reacts or associates more frequently, more rapidly, longer in duration, and/or with higher affinity than it reacts or associates with a particular antigen or cell expressing it. For example, the compound binds to CD131 with substantially greater affinity (e.g., 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than other cytokine receptors or with antigens that are normally recognized by multi-reactive natural antibodies (i.e., recognized by naturally occurring antibodies that are known to bind to a variety of antigens naturally occurring in humans). Generally, but not necessarily, reference to binding means specific binding, and each term should be understood to provide explicit support for the other term.

If the protein or antibody is cleaved from another polypeptide with a smaller dissociation constant (K _D ) K of (2) _D Binding to a polypeptide, the protein or antibody is said to "preferentially bind" to the polypeptide. In one embodiment, if the protein or antibody is specific for K of another polypeptide than the protein or antibody _D An affinity of at least about 20-fold or 40-fold or 60-fold or 80-fold or 100-fold or 120-fold or 140-fold or 160-fold (i.e., K) _D ) Binding to a polypeptide, the protein or antibody is said to preferentially bind to the polypeptide.

For clarification purposes, and as based on the illustrations hereinExemplary subject matter, it will be apparent to the skilled artisan that references to "affinity" in this specification refer to K for a protein or antibody _D 。

For clarification purposes, and as will be apparent to the skilled artisan based on the description herein, reference is made to "K of X nM or less _D "to be understood as meaning K _D Is equal to x nM, or lower in value. As the skilled person will appreciate, K _D Corresponds to a higher (i.e., stronger) affinity, i.e., 2nM has an affinity that is stronger than 3 nM.

"IC of at least about … … _a0 "to be understood as meaning IC ₅₀ Equal to the value or greater (i.e., when IC ₅₀ Lower, enumerated values), i.e., 2nM IC ₅₀ IC greater than 3nM ₅₀ . In other words, this term may be "X" or a smaller IC ₅₀ ", wherein X is a value described herein.

As used herein, the term "epitope" (synonymous "antigenic determinant") is understood to mean a region of CD131 to which a protein comprising the antigen binding site of an antibody binds. This term is not necessarily limited to the particular residue or structure with which the protein is contacted. For example, this term includes the region spanning the amino acids to which the protein is exposed and/or 5-10 or 2-5 or 1-3 amino acids outside of this region. In some embodiments, an epitope comprises a series of discrete amino acids, i.e., a "conformational epitope," that are positioned close to each other when CD131 is folded. The skilled artisan will also appreciate that the term "epitope" is not limited to a peptide or polypeptide. For example, the term "epitope" includes chemically active surface groups of a molecule, such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and in certain embodiments may have specific three-dimensional structural features and/or specific charge features.

The term "competitively inhibit" is understood to mean that the protein of the present disclosure (or antigen binding site thereof) reduces or prevents binding of the antibody or protein to CD131. This may be due to the binding of the protein (or antigen binding site) and the antibody to the same or overlapping epitopes. It will be apparent from the foregoing that the protein need not completely inhibit binding of the antibody, but need only reduce binding by a statistically significant amount, e.g., by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the protein reduces binding of the antibody by at least about 30%, more preferably by at least about 50%, more preferably by at least about 70%, still more preferably by at least about 75%, even more preferably by at least about 80% or 85%, and even more preferably by at least about 90%. Methods for determining competitive inhibition binding are known in the art and/or described herein. For example, the antibody is exposed to CD131 in the presence or absence of a protein. Proteins are considered to competitively inhibit antibody binding if less antibody is bound in the presence of the protein than in the absence of the protein. In one embodiment, competitive inhibition is not due to steric hindrance.

In the context of two epitopes, "overlapping" should be understood to mean that the two epitopes share a sufficient number of amino acid residues to allow a protein (or antigen binding site thereof) that binds to one epitope to competitively inhibit a protein (or antigen binding site) that binds to the other epitope. For example, an "overlapping" epitope shares at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 amino acids.

Acute Respiratory Distress Syndrome (ARDS)

The present disclosure provides, for example, a method for treating or preventing Acute Respiratory Distress Syndrome (ARDS) in a subject.

ARDS is a life-threatening condition characterized by bilateral pulmonary infiltration, severe hypoxia, and non-cardiac pulmonary edema. ARDS causes severe lung injury and 30-50% of the attributed mortality (attributable mortality rate). To date, there is no effective pharmacotherapy. Infectious causes, such as sepsis and pneumonia (including influenza and coronavirus infections), are the primary causes of ARDS. Thus, treatment of these underlying diseases or conditions (e.g., infections) is also contemplated in combination with the methods of the present disclosure.

Histologically, ARDS in humans is characterized by severe acute inflammatory response and neutrophilic alveolitis in the lungs. Inflammatory stimuli such as endotoxins (lipopolysaccharide, LPS) from microbial pathogens are well accepted for their ability to induce pulmonary inflammation, and systemic and intratracheal experimental administration of LPS has been used to induce pulmonary inflammation in animal models of ARDS, as described herein. LPS acts via Toll-like receptor 4 (TLR 4) to increase the expression of inflammatory cytokines and chemokines and up-regulate leukocyte adhesion molecules, leading to endothelial cell activation.

The physiological hallmark of ARDS is the disruption of the alveolar-capillary membrane barrier (i.e., pulmonary vascular leakage), leading to the development of non-cardiac pulmonary edema, in which protein exudates flood the alveolar space, impair gas exchange, and lead to respiratory failure. Alveolar epithelial and endothelial cell injury and/or death are both associated with the pathogenesis of ARDS. ARDS remains a significant cause of long-term mechanical ventilation in the Intensive Care Unit (ICU), and the ARDS-related mortality rates are as high as 30-50% despite optimal ICU support therapy.

ARDS was defined by the expert panel in 2012 as a advocate by the Berlin definition (european intensive care medical society (European Society ofIntensive Care Medicine) approved by the american society of thoracic and intensive care medical society (American Thoracic Society and the Society of Critical Care Medicine)). Currently there are three phases: mild, moderate and severe, with associated mortality increasing (27%, 95% ci,24% -30%, 32%, 95% ci,29% -34%, and 45%, 95% ci,42% -48%, p < 0.001), respectively); and the median duration of mechanical ventilation increased in survivors (5 days; quartile range [ IQR ],2-11;7 days; IQR,4-14; and 9 days; IQR,5-17; p < 0.001), respectively. The definition was empirically evaluated using a patient-level meta analysis of 4188 patients with ARDS from 4 multi-center clinical datasets and 269 patients with ARDS from 3 single-center datasets containing physiological information.

According to Berlin definition, ARDS is defined according to the following:

(1) Occurs within 1 week of onset of clinical injury or respiratory symptoms;

(2) Acute hypoxemic respiratory failure, such as by passing PaO 300mmHg or less under Continuous Positive Airway Pressure (CPAP) or Positive End Expiratory Pressure (PEEP) of at least 5cm ₂ /FiO ₂ Ratio-determined, where PaO ₂ Is the partial pressure of oxygen in arterial blood, and FiO ₂ Is the fraction of inhaled oxygen;

(3) Bilateral shadows on chest radiographs, which cannot be explained entirely by effusion, solid changes, or lung tension; and

(4) Respiratory failure, which cannot be explained entirely by heart failure or excessive body fluid.

In one embodiment of the methods of the present disclosure, the subject meets Berlin criteria of ARDS described above. In other embodiments, the subject may not yet meet Berlin standards for ARDS, but is determined to be at risk of developing ARDS. Such subjects may be administered a compound that binds to CD131 to prevent the onset of ARDS.

As used herein, the term "at risk" means that the subject has an increased chance of developing ARDS as compared to a normal individual. The subject may be identified as being at risk of developing ARDS using any method known in the art. For example, if a subject has a common underlying cause of ARDS (e.g., sepsis, pneumonia, trauma, etc.) and has respiratory symptoms, such as shortness of breath and/or shortness of breath, the subject may be identified as being at risk of developing ARDS. Other methods suitable for identifying subjects at risk of developing ARDS include, for example, WO2018/204509; luo et al, 2017, J Thorac J.disease, 9, 3979-3995; de Haro et al, 2013, annual intensive care (Annals of Intensive Care) 3, 11; iriyama et al 2020, journal of intensive care (Journal of Intensive Care), 8,7; gajic et al, 2011, journal of respiratory and critical care medicine (Am J Respir Crit CareMed), 183, 462-470; and Yadav et al, 2017, journal of respiratory and critical care medicine, 195, 725-736.

The severity of ARDS can be categorized as follows:

(1) Mild ARDS: paO (PaO) ₂ /FiO ₂ 200-300mmHg;

(2) Moderate ARDS: paO (PaO) ₂ /Fi0 ₂ 100-200mmHg; and

(3) Severe ARDS: paO (PaO) ₂ /FiO ₂ Less than or equal to 100mmHg.

In some embodiments of the methods of the present disclosure, the ARDS is mild ARDS. In some embodiments, the ARDS is a moderate ARDS. In some embodiments, the ARDS is a severe ARDS.

The method of the present disclosure is applicable to all reasons of ARDS. The most common causes of ARDS are sepsis, inhaled noxious substances, pneumonia, severe wounds (bilateral lung contusions, fat embolism after long bone fractures, sepsis occurring several days after severe wounds or burns and extensive traumatic tissue injury), extensive blood transfusion, transfusion-related acute lung injuries, lung and hematopoietic stem cell transplants, other acute inflammatory diseases, drugs and alcohol, and mutations in genetic determinants such as the surface active protein B (SP-B) gene.

More recently, ARDS has been shown to be caused by severe coronavirus disease 2019 (covd-19), viral pneumonia from SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection. The methods of the present disclosure may be used to treat or prevent ARDS in subjects with severe covd-19. Thus, in some embodiments, the present disclosure provides a method of treating covd-19 in a subject, the method comprising administering to the subject a compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF. Common symptoms of covd-19 include fever, coughing, fatigue, shortness of breath, and loss of sense of smell and taste. Although most cases result in mild symptoms, some develop ARDS. Other coronavirus infections have in the past led to SARS and MERS, which may also lead to ARDS. For example, SARS-CoV-2 infection can be confirmed by positive detection of viral RNA in nasopharyngeal secretions using a specific PCR assay. Covd-19 disease can be demonstrated by consistent clinical history, epidemiological exposure, and positive SARS-CoV-2 assays. ARDS associated with COVID-19 may be diagnosed when a subject with a confirmed infection with COVID-19 meets the Berlin ARDS diagnostic criteria described above.

Antibodies to

In one embodiment, the compound described herein according to any embodiment is a protein comprising an antigen binding site of an antibody. In some embodiments, the compound is an antibody.

Methods for producing antibodies are known in the art and/or are described in Harlow and Lane (eds.) [ antibodies: the laboratory Manual is described in Cold spring harbor laboratory (1988). Typically, in such methods, CD131 or a region thereof (e.g., an extracellular domain) or an immunogenic fragment or epitope thereof or a cell expressing and displaying it (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, e.g., a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

Monoclonal antibodies are one exemplary form of antibodies contemplated by the present disclosure. The term "monoclonal antibody" or "mAb" refers to a homogeneous population of antibodies capable of binding to the same antigen (e.g., binding to the same epitope within an antigen). This term is not intended to limit the source of the antibody or the manner in which it is made.

For the production of mAbs, any of a number of known techniques may be used, such as for example the procedure illustrated in US4196265 or Harlow and Lane (1988) (supra).

Alternatively, ABL-MYC technology (NeoClone, madison, wis., 53713, U.S.) was used to generate MAbs-secreting cell lines (e.g., as described in Largaaespada et al J.Immunol. Methods, 197:85-95, 1996).

Antibodies may also be generated or isolated by screening a display library (e.g., a phage display library), e.g., as described in US6300064 and/or US 5885793. For example, the inventors of the present invention have isolated fully human antibodies from phage display libraries.

The antibodies of the present disclosure may be synthetic antibodies. For example, the antibody is a chimeric, humanized, human or deimmunized antibody.

In one embodiment, the antibodies described herein are chimeric antibodies. The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species (e.g., primate, such as human) or belonging to another antibody class or subclass. Methods for producing chimeric antibodies are described, for example, in US 4817567 and US 5807715.

Antibodies of the disclosure may be humanized or human.

The term "humanized antibody" is understood to mean a subclass of chimeric antibodies having antigen binding sites or variable regions derived from antibodies from non-human species as well as residual antibody structures based on the structure and/or sequence of human antibodies. In humanized antibodies, the antigen binding site will typically comprise Complementarity Determining Regions (CDRs) from a non-human antibody grafted onto the appropriate FR in the variable region of a human antibody and the remaining regions from the human antibody. The antigen binding site may be wild-type (i.e., identical to the antigen binding site of a non-human antibody) or modified by one or more amino acid substitutions. In some cases, FR residues of a human antibody are substituted with corresponding non-human residues.

Methods for humanizing non-human antibodies or portions thereof (e.g., variable regions) are known in the art. Humanization may be performed according to the methods of US5225539 or US 5585089. Other methods for humanizing antibodies are not excluded.

The term "human antibody" as used herein refers to a polypeptide having a variable region (e.g., V _H 、V _L ) And optionally a constant region derived from or corresponding to a sequence found in a human (e.g., in a human germline or somatic cell).

Exemplary human antibodies are described herein and include 9A2-VR24.29 (also referred to as "CSL 311") described in WO 2017/088028 and Sun et al (1999) blood, 94:1943-1951 and/or a protein comprising a variable region thereof or a derivative thereof. These human antibodies offer the advantage of reduced immunogenicity in humans compared to non-human antibodies.

In one embodiment, the antibody is a multispecific antibody. For example, a compound that binds to CD131 may be a protein that comprises an antigen binding site that binds to CD131 and an additional antigen binding site that binds to a different antigen. Thus, in some embodiments, the antibody is a bispecific antibody.

Proteins containing antibody binding domains

Single domain antibodies

In some embodiments, the compounds of the present disclosure are proteins that are or comprise single domain antibodies (which may be used interchangeably with the terms "domain antibody" or "dAb" or "nanobody"). Single domain antibodies are antibody fragments consisting of a single monomer variable antibody domain. Like whole antibodies, it is capable of selectively binding to a specific antigen. In the case of molecular weights of only 12-15kDa, single domain antibodies are much smaller than common antibodies consisting of two heavy protein chains and two light chains (150-160 kDa), and even smaller than Fab fragments (about 50kDa, one light chain and half heavy chain) and single chain variable fragments (about 25kDa, two variable domains, one from the light chain and one from the heavy chain). In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, inc., waltham, mass.; see, e.g., US 6248516).

In some embodiments, the single domain antibody is V _H H fragment. V (V) _H The H fragment consists of the variable domain of a camelid heavy chain antibody (V _H ) The composition is as follows.

In some embodiments, the single domain antibody is V _NAR Fragments. V (V) _NAR Fragments are composed of the variable domains of heavy chain antibodies from cartilaginous fish (V _H ) The composition is as follows.

Double, triple and quadruple bodies

In some embodiments, the proteins of the present disclosure are or comprise binary, trisomy, tetrasomy, or higher order protein complexes, such as those described in WO98/044001 and/or WO 94/007921.

Single chain Fv (scFv)

The skilled artisan will appreciate that scFv comprises V in a single polypeptide chain _H And V _L Zone and at V _H And V _L A polypeptide linker therebetween, said polypeptide linker allowing scF _v Capable of forming the structure required for antigen binding (i.e., V of a single polypeptide chain _H And V _L Associated with each other to form Fv). For example, the linker comprises more than 12 amino acid residues, wherein (Gly ₄ Ser) ₃ Is one of the more advantageous linkers for scFv.

Heavy chain antibodies

Heavy chain antibodies differ in structure from many other forms of antibodies, so long as they contain a heavy chain but not a light chain. Thus, these antibodies are also referred to as "heavy chain only antibodies". Heavy chain antibodies are found in, for example, camelids and cartilaginous fish (also known as IgNAR).

A general description of heavy chain antibodies and their variable regions from camelids and methods for their production and/or isolation and/or use is found in particular in the following references WO94/04678, WO 97/49505 and WO 97/49505.

A general description of heavy chain antibodies and variable regions thereof to cartilaginous fish and methods of producing and/or isolating and/or using the same is found, inter alia, in WO 2005/118629.

Other antibodies and antibody fragments

Other antibodies and antibody fragments are also contemplated by the present disclosure, such as:

(i) A "key and hole" bispecific protein as described in US5,731,168;

(ii) Heterologous conjugate proteins (heteroconjugate protein), for example as described in US4,676,980;

(iii) Heterologous conjugate proteins produced using chemical cross-linking agents, for example, as described in US4,676,980; and

(iv)Fab ₃ (e.g. as described in EP 19930302894).

V-like proteins

An example of a compound of the present disclosure is a T cell receptor. T cell receptors have two V domains that combine to form a structure similar to the Fv module of an antibody. Novotny et al, journal of the national academy of sciences (Proc Natl Acad Sci USA), 88:8646-8650, 1991 describes how two V domains of T cell receptors (called α and β) can be fused and expressed as single chain polypeptides, and how surface residues can be altered to reduce direct analogy to antibody scF _v Is a hydrophobic property of (a). Other publications describing the generation of single-chain T cell receptors or multimeric T cell receptors comprising two V- α and V- β domains include WO1999/045110 or WO2011/107595.

Other non-antibody proteins comprising antigen binding domains include proteins having V-like domains, which are typically monomeric. Examples of proteins comprising such V-like domains include CTLA-4, CD28 and ICOS. Additional disclosures of proteins comprising such V-like domains are included in WO 1999/045110.

Adnectin

In one embodiment, the compound of the present disclosure is adnectin. Tenth fibronectin type III based on human fibronectin ¹⁰ Fn 3) domain, wherein the loop region is altered to confer antigen binding. For example, in ¹⁰ Three loops at one end of the β -sandwich of Fn3 domains can be engineered to enable adnectin to specifically recognize an antigen. For further details, see US20080139791 or WO2005/056764.

Anti-carrier protein (Anticalin)

In further embodiments, the compounds of the present disclosure are anti-cargo proteins. Anti-cargo proteins are derived from lipocalins, a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilirubin, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with multiple loops at the open end of the cone structure, which can be engineered to bind antigens. Such engineered lipocalins are known as anti-calins. For further description of anti-transporters, see US7250297B1 or US20070224633.

Affinity body

In further embodiments, the compounds of the present disclosure are affibodies. An affibody is a scaffold derived from the Z domain (antigen binding domain) of protein a of staphylococcus aureus (Staphylococcus aureus), which can be engineered to bind to an antigen. The Z domain consists of a triple helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. See EP1641818 for further details.

Avimer

In further embodiments, the compound of the present disclosure is Avimer. Avimer is a multidomain protein derived from the a-domain scaffold family. The native domain of about 35 amino acids adopts a defined disulfide structure. Diversity results from shuffling of the natural variations exhibited by the a domain family. See WO2002/088171 for additional details.

DARPin

In further embodiments, the compounds of the present disclosure are engineered ankyrin repeat proteins (DARPin). DARPin is derived from an ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and one beta turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha helix and beta turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). See US20040132028 for further details.

Deimmunized proteins

The present disclosure also contemplates deimmunized antibodies or proteins. Deimmunized antibodies and proteins have one or more epitopes, such as B cell epitopes or T cell epitopes, removed (i.e., mutated) thereby reducing the likelihood that a mammal will mount an immune response against the antibody or protein. Methods for producing deimmunized antibodies and proteins are known in the art and are described, for example, in WO2000/34317, WO2004/108158 and WO 2004/064724.

Methods for introducing suitable mutations and expressing and assaying the resulting proteins will be apparent to the skilled artisan based on the description herein.

Protein mutation

The present disclosure also contemplates mutant forms of the proteins of the present disclosure. For example, such muteins comprise one or more conservative amino acid substitutions compared to the sequences shown herein. In some embodiments, the protein comprises 30 or less or 20 or less or 10 or less, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which the amino acid residue is substituted with an amino acid residue having similar side chains and/or hydrophilicity (hydrophilicity) and/or affinity (hydropathicity).

In one embodiment, the mutein has only or no more than one or two or three or four or five or six conservative amino acid changes when compared to the naturally occurring protein. Details of conservative amino acid changes are provided below. As the skilled artisan will appreciate, for example, from the disclosure herein, it is reasonable to predict that such minor changes will not alter the activity of the protein.

Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The present disclosure also contemplates non-conservative amino acid changes (e.g., substitutions) in a protein of the disclosure, e.g., in a CDR (e.g., CDR 3). In one embodiment, the protein comprises fewer than 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions, e.g., in CDR3 (as in CDR 3).

The present disclosure also contemplates one or more insertions or deletions compared to the sequences described herein. In some embodiments, the protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 insertions and/or deletions.

Constant region

The present disclosure encompasses proteins and/or antibodies described herein comprising the constant regions of antibodies. This includes antigen binding fragments of antibodies fused to Fc.

Sequences useful for producing the constant regions of the proteins of the present disclosure can be obtained from a number of different sources. In some embodiments, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, igG, igD, igA and IgE, and any antibody isotype, including IgG1, igG2, igG3, and IgG4. In one embodiment, the constant region is a human isotype IgG4 or a stabilized IgG4 constant region.

In one embodiment, the Fc region of the constant region has a reduced ability to induce effector function, e.g., as compared to a native or wild-type human IgG1 or IgG3 Fc region. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of a protein containing an Fc region are known in the art and/or described herein.

In one embodiment, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), such as a human IgG4 Fc region. The sequence of a suitable IgG4 Fc region will be apparent to the skilled artisan and/or may be obtained from publicly available databases (e.g., available from national center for biotechnology information (National Center for Biotechnology Information).

In one embodiment, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce Fab arm exchange or to undergo Fab arm exchange or a tendency to form half antibodies. "Fab arm exchange" refers to a protein modification to human IgG4 in which one IgG4 heavy chain and attached light chain (half molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can obtain two different Fab arms (resulting in a bispecific molecule) that recognize two different antigens. Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. An "half antibody" is formed when an IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

In one embodiment, the stabilized IgG4 constant region comprises proline at position 241 of the hinge region of the system according to Kabat (Kabat et al, sequences of proteins of immunological interest, washington, d.c., U.S. health and human service, 1987 and/or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al, "sequence of proteins of immunological interest", washington, division of service, U.S. health and human, 2001 and Edelman et al, proc. Natl. Acad. Sci. USA, 63, 78-85, 1969). In human IgG ₄ In general, such residues are serine. After serine has replaced proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, the skilled artisan will appreciate that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which imparts fluidity to the two Fab arms of the antibody. The hinge region includes cysteine residues that participate in the disulfide bond between the heavy chains. According to the numbering system of Kabat, it is generally defined as the extension from Glu226 to Pro243 of human IgG 1. The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter-heavy chain disulfide bond (S-S) in the same position (see, e.g., WO 2010080538).

Further examples of stabilized IgG4 antibodies are antibodies in which arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine or leucine (e.g. as described in WO 2006/033386). The Fc region of the constant region may additionally or alternatively comprise a residue at a position corresponding to 405 (according to the EU numbering system) selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine. Optionally, the hinge region comprises a proline (i.e., CPPC sequence) at position 241 (as described above).

In another embodiment, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region is an IgG1 Fc region comprising substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another embodiment, the Fc region is an IgG1 Fc region comprising one or more of the following alterations: deletions of E233P, L234V, L235A and G236, and/or one or more of the following changes: A327G, A S and P331S (Armour et al, european journal of immunology (Eur J immunol.)) 29:2613-2624, 1999; shields et al, J Biol chem.), 276 (9): 6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall' Acqua et al, journal of immunology (J immunol.), 177:1129-11382006; and/or Hezareh journal of virology (J Virol); 75:12161-12168, 2001.

In another embodiment, the Fc region is a chimeric Fc region, e.g., comprising at least one C from an IgG4 antibody _H 2 domain and at least one C from an IgG1 antibody _H 3, wherein the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO 2010/085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

Additional modifications

The present disclosure also contemplates additional modifications to the antibodies or proteins of the present disclosure.

For example, an antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, an antibody comprises an Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region for a neonatal Fc receptor (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH (e.g., about pH 6.0) to promote Fc/FcRn binding in the endosome. In one embodiment, the Fc region has an increased affinity for FcRn at about pH 6 as compared to the affinity of the Fc region at about pH 7.4, which facilitates the re-release of Fc into the blood after cell recirculation. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L or T252A, T S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US 7083784.

The protein may be a fusion protein. Thus, in one embodiment, the protein further comprises albumin, a functional fragment or variant thereof. In one embodiment, the albumin, functional fragment or variant thereof is serum albumin, such as human serum albumin. In one embodiment, the albumin, functional fragment or variant thereof comprises one or more amino acid substitutions, deletions or insertions, e.g., no more than 5 or 4 or 3 or 2 or 1 substitutions. Amino acid substitutions suitable for use in the present disclosure will be apparent to the skilled person and include naturally occurring substitutions and engineered substitutions, for example, as those described in WO2011/051489, WO2014/072481, WO2011/103076, WO2012/112188, WO2013/075066, WO2015/063611 and WO 2014/179657.

In one embodiment, the proteins of the present disclosure additionally comprise a soluble complement receptor or a functional fragment or variant thereof. In one embodiment, the protein further comprises a complement inhibitor.

Protein production

In one embodiment, a protein described herein according to any embodiment is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and/or known in the art.

Recombinant expression

In another embodiment, the protein described herein according to any embodiment is recombinant.

In the case of recombinant proteins, the nucleic acid encoding the protein may be cloned into an expression construct or vector, which is then transfected into a host cell, such as an E.coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a simian COS cell, a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a myeloma cell that does not otherwise produce the protein. Exemplary cells for expressing the protein are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these objectives are known in the art and are described, for example, in Ausubel et al (eds.), "Current protocols for molecular biology," Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al, "molecular cloning: laboratory Manual, cold spring harbor laboratory Press (1989). A variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art, see for example US 481657 or US5530101.

After isolation, the nucleic acid is operably inserted into a promoter linked to an expression construct or expression vector for further cloning (amplification of DNA) or for expression in a cell-free system or in a cell.

As used herein, the term "promoter" is to be considered in its broadest context and includes transcriptional regulatory sequences of genomic genes, including TATA boxes or initiation elements (initiator element) required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter expression of a nucleic acid, for example, in response to developmental and/or external stimuli or in a tissue-specific manner. The term "promoter" is also used herein to describe a recombinant, synthetic or fused nucleic acid, or derivative that confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter spatial and/or temporal expression of the nucleic acid.

As used herein, the term "operably linked to … …" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is under the control of the promoter.

Many vectors for expression in cells are available. The carrier component generally includes, but is not limited to, one or more of the following: signal sequences, protein-encoding sequences (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. The skilled artisan will know the appropriate sequences for expression of the protein. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, ipp, or thermostable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signals).

Exemplary promoters active in mammalian cells include the cytomegalovirus very early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF 1), the micronuclear RNA promoter (U1 a and U1 b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV 40), the rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter; hybrid regulatory elements comprising a CMV enhancer/β -actin promoter or an immunoglobulin promoter or active fragment thereof. An example of a useful mammalian host cell line is the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells, subclones for growth in suspension culture, baby hamster kidney cells (BHK, ATCC CCL 10), or Chinese hamster ovary Cells (CHO).

Typical promoters suitable for expression in yeast cells such as, for example, yeast cells selected from the group consisting of Pichia pastoris, saccharomyces cerevisiae (Saccharomyces cerevisiae) and schizosaccharomyces pombe (s.pombe) include, but are not limited to, the ADH1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

Means for introducing an isolated nucleic acid or an expression construct comprising said nucleic acid into a cell for expression are known to the person skilled in the art. The technique used for a given cell depends on known successful techniques. Methods for introducing recombinant DNA into cells include microinjection; transfection mediated by DEAE-dextran; liposome-mediated transfection, such as by use of lipofectamine (Gibco, maryland, usa) and/or cellfectin (Gibco, maryland, usa); PEG-mediated DNA uptake; electroporation and microprojectile bombardment, such as by using DNA coated tungsten or gold particles (Agracetus, wisconsin, U.S.A.), among others.

Host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F (Sigma), minimal essential media ((MEM), sigma), RPMl-1640 (Sigma) and Du's modified Igor medium ((DMEM), sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

Separation of proteins

Methods for isolating proteins are known in the art and/or described herein.

In the case where the protein is secreted into the culture medium, the supernatant from such an expression system may first be concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants. Alternatively or additionally, the supernatant may be filtered and/or separated from the cells expressing the protein, for example using continuous centrifugation.

Proteins produced by the cells may be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein a affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and are described, for example, in WO1999/57134 or Ed Harlow and David Lane (eds.) "antibodies: a description is given in the laboratory Manual, cold spring harbor laboratory (1988).

Those skilled in the art will also appreciate that the protein may be modified to include a tag that facilitates purification or detection, such as a polyhistidine tag, for example, a hexahistidine tag, or an influenza virus Hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein comprising a hexahistidine tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively or additionally, a ligand or antibody that binds to the tag is used in the affinity purification method.

Nucleic acid compounds that bind to CD131

In one embodiment, the compound that binds to CD131 is a nucleic acid aptamer (an adaptive oligomer). An aptamer is a single-stranded oligonucleotide or oligonucleotide analog capable of forming a secondary and/or tertiary structure that provides the ability to bind to a particular target molecule, such as a protein or small molecule, e.g., CD131. Thus, an aptamer is an oligonucleotide similar to an antibody. Typically, the aptamer comprises from about 15 to about 100 nucleotides, such as from about 15 to about 40 nucleotides, for example from about 20 to about 40 nucleotides, as oligonucleotides having a length falling within these ranges can be prepared by conventional techniques.

The aptamer may be isolated or identified from a library of aptamers. For example, by cloning random oligonucleotides into a vector (or expression vector in the case of RNA aptamers) to generate an aptamer library, wherein the random sequences are flanked by known sequences that provide binding sites for PCR primers. An aptamer that provides a desired biological activity (e.g., specifically binds to CD 131) is selected. For example, exponential enrichment ligand systematic evolution (SELEX) was used to select for aptamers with increased activity. Suitable methods for generating and/or screening aptamer libraries are described, for example, in ellomington and szostank, nature 346:818-22, 1990; US 5270163 and/or US 5475096.

Determination of Activity of Compounds

Binding to CD131

Methods for assessing binding of candidate compounds to proteins (e.g., CD 131) are known in the art, for example as described in scens (protein purification: principle and practice (Protein purification: principles and practice), third edition, springer Verlag, 1994). Such methods typically involve labeling the protein and contacting it with an immobilized compound. After washing to remove non-specifically bound proteins, the amount of label is detected, thereby detecting the amount of bound protein. Of course, the protein may be immobilized and the compound may be labeled. Panning type assays may also be used. Alternatively or additionally, surface plasmon resonance assays may be used. The level of binding can also be conveniently determined using a biosensor.

Optionally, determining the dissociation constant (Kd) of the compound for CD131 or an epitope thereof. In one embodiment, the "Kd" or "Kd value" of a compound that binds to CD131 is measured by a radiolabeled or fluorescent labeled CD131 binding assay. This assay equilibrates the compound with a minimum concentration of labeled CD131 in the presence of unlabeled CD131 of the titration series. After washing to remove unbound CD131, the amount of label is determined, which is indicative of the Kd of the protein.

According to another embodiment, kd or Kd values are measured using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore corporation, piscataway, NJ) with immobilized CD131 or regions thereof.

Epitope mapping

In another embodiment, epitopes bound by the proteins described herein are plotted. Epitope mapping methods will be apparent to the skilled artisan. For example, a series of overlapping peptides, e.g., peptides comprising 10-15 amino acids, spanning the CD131 sequence or a region thereof comprising an epitope of interest are generated. The protein is then contacted with each peptide and the peptide to which it binds is determined. This allows the determination of peptides comprising epitopes to which the protein binds. If multiple discrete peptides are bound by a protein, the protein may bind a conformational epitope.

Alternatively or additionally, amino acid residues within CD131 are mutated, e.g., by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are identified. Any mutation that reduces or prevents protein binding may be within the epitope bound by the protein.

Additional methods are exemplified herein, and involve binding CD131 or a region thereof to the immobilized proteins of the disclosure, and digesting the resulting complexes with a protease. Peptides that remain bound to the immobilized protein are then isolated and analyzed, for example, using mass spectrometry, to determine their sequence.

Additional methods involve converting hydrogen in CD131 or a region thereof to deuterium and binding the resulting protein to the immobilized protein of the present disclosure. Deuterium is then converted back to hydrogen, CD131 or regions thereof are isolated, digested with enzymes and analyzed, e.g., using mass spectrometry, to identify those regions containing deuterium that are protected from conversion to hydrogen by binding to the proteins described herein.

Alternatively, the epitope bound by the protein may be determined by X-ray crystallography. For example, a complex is formed between the protein and CD131, and then crystallized. The resulting crystals are then subjected to x-ray diffraction analysis to determine the atomic coordinates of the amino acids in the complex. The epitope comprises the amino acid in CD131 that is in contact with the protein, according to atomic coordinates determined by x-ray diffraction.

Determination of competitive binding

The assay for determining proteins that competitively inhibit the binding of antibody 9A2-VR24.29 will be apparent to the skilled artisan. For example, 9A2-VR24.29 is conjugated to a detectable label, such as a fluorescent label or a radioactive label. The labeled antibodies and test proteins are then mixed and contacted with CD131 or a region thereof (e.g., a polypeptide comprising SEQ ID NO:1 or 5) or cells expressing the same. The level of labeled 9A2-VR24.29 is then determined and compared to the level determined when the labeled antibody is contacted with CD131, a region or a cell in the absence of protein. If the level of labeled 9A2-VR24.29 is reduced in the presence of the test protein as compared to in the absence of the protein, the protein is said to competitively inhibit the binding of 9A2-VR24.29 to CD 131.

Optionally, the test protein is conjugated to 9A2-VR24.29 with a different label. Such surrogate markers allow for the detection of the binding level of the test protein to CD131 or a region or cell thereof.

In another embodiment, the protein is allowed to bind to CD13l or a region thereof (e.g., a polypeptide comprising SEQ ID NO:1 or 5) or a cell expressing it, prior to contacting CD131, the region or the cell with 9A2-VR24.29. The reduced amount of 9A2-VR24.29 bound in the presence of protein as compared to in the absence of protein indicates that the protein competitively inhibits the binding of 9A2-VR24.29 to CD 131. The mutual assay can also be performed using a labeled protein and first allowing 9A2-VR24.29 to bind to CD 131. In this case, a decrease in the amount of labeled protein bound to CD131 in the presence of 9A2-VR24.29 as compared to in the absence of 9A2-VR24.29 indicates that the protein competitively inhibits the binding of 9A2-VR24.29 to CD 131.

Mutant forms of CD131 and/or SEQ ID NO:1 or 5 and/or a ligand binding domain of CD131 that binds to 9A2-VR24.29, e.g., as described herein.

Determining neutralization

In one embodiment, the compound that binds to CD131 reduces or prevents binding of IL-3, IL-5, and/or GM-CSF to a receptor comprising CD131 (e.g., IL-3R, IL-5R and/or GM-CSF-R, respectively). These assays can be performed as competitive binding assays using labeled IL-3/I1-5/GM-CSF and/or labeled compounds. For example, cells expressing the relevant receptor are brought into the presence of IL-3/IL-5/GM-CSF Or in the absence of a CD131 binding compound and detecting the amount of bound label. A decrease in the amount of label bound in the presence of the CD131 binding compound as compared to the absence of the CD131 binding compound indicates that the compound decreases or prevents binding of IL-3/I1-5/GM-CSF to a receptor comprising CD 131. Determination of IC by testing various concentrations of compounds ₅₀ I.e. the concentration of a compound that reduces the amount of IL-3/11-5/GM-CSF that binds to a receptor comprising CD131, or the EC can be determined ₅₀ I.e.a protein concentration of 50% of the maximum inhibition of IL-3/1l-5/GM-CSF binding to CD131 by the compound is achieved.

In further embodiments, the CD131 binding compound reduces or prevents IL-3/O1-5/GM-CSF mediated proliferation of leukemia cell line TF-1. For example, TF-1 cells are cultured in the absence of IL-3/11-5/GM-CSF for a time sufficient to stop them from proliferating (e.g., 24-48 hours). The cells are then cultured in the presence of IL-3/IL-5/GM-CSF and various concentrations of CD 131-binding compound. Control cells were not contacted with either compound (positive control) or IL-3/IL-5/GM-CSF (negative control). And then using standard techniques (e.g ³ H-thymidine incorporation) to assess cell proliferation. CD131 binding compounds that reduce or prevent cell proliferation to levels lower than the positive control in the presence of IL-3 are believed to neutralize IL-3 signaling. Determination of IC by testing various concentrations of CD131 binding compound ₅₀ 。

In another embodiment, the CD131 binding compound inhibits or prevents STAT-5 activation. For example, cells (e.g., TF-1 cells) comprising a beta-lactamase reporter gene under the control of an interferon regulatory factor 1 (irf 1) response element in the presence of IL-3 and/or GM-CSF. Suitable cells are available from life technologies company (Life Technologies Corporation). The cells are also contacted with a suitable substrate (e.g., a negatively charged fluorescent β -lactamase substrate, such as CCF2 or CCF 4) and the change in signal (e.g., fluorescence) is determined. A decrease in signal change in the positive control (i.e., cells contacted with IL-3 and/or GM-CSF in the absence of the compound) indicates that the compound reduces or prevents IL-3 and/or GM-CSF-induced STAT-5 signaling.

In further embodiments, the CD131 binding compounds of the present disclosure affect immune cells. For example, the CD131 binding compound reduces or inhibits activation of isolated human neutrophils by GM-CSF as determined by reducing or inhibiting GM-CSF-induced increase in neutrophil cell size. For example, neutrophils (e.g., about 1X 10 in the presence of CD131 binding protein and GM-CSF ⁵ Individual cells) for a suitable time (e.g., about 24 hours). Cells are then fixed (e.g., with formaldehyde) and analyzed for forward scatter using flow cytometry.

In one embodiment, the CD131 binding compound reduces or inhibits IL-8 secretion induced by IL-3 of human basophils. For example, basophils (e.g., about 1X 10 ⁵ Individual cells) in the presence of a CD131 binding compound and IL-3 for a suitable time (e.g., 24 hours). Then for example using ELISA (e.g. available from R&Obtained by D systems) to assess IL-8 secretion.

In further embodiments, the CD131 binding compound reduces or prevents IL-3 mediated survival or pDC. For example, pDC is cultured in the presence of CD131 binding compound and IL-3 for a suitable time (e.g., 24 hours). Cell survival can then be assessed, for example, using standard methods (e.g., the ViaLight Plus kit from Lonza).

In further embodiments, the CD131 binding compound reduces or prevents activation of human peripheral blood eosinophils by IL-5 as determined by assessing changes in forward scatter assessed by flow cytometry. For example, eosinophils (e.g., about 1X 10 ⁵ Individual cells) are cultured in the presence of a CD131 binding compound and IL-5 for a suitable time (e.g., about 24 hours). Cells are then fixed (e.g., in formaldehyde) and the change in forward scatter is assessed, for example, using flow cytometry.

In further embodiments, the CD131 binding compounds of the present disclosure reduce or prevent survival of human peripheral blood eosinophils in the presence of IL-5 and/or GM-CSF and/or IL-3. For example, eosinophils in the presence of CD131 binding compounds and IL-5 and/or GM-CSF and/or IL-3(e.g., about 1X 10) ⁴ The cells) are cultured for a suitable time (e.g., about 5 days) and the number of cells is assessed using standard methods (e.g., the Vialight Plus kit from Lonza).

In still further embodiments, the CD131 binding compounds of the present disclosure reduce or prevent IL-3 induced TNF alpha release from human mast cells. For example, human cultured mast cells (e.g., ten week old peripheral blood derived cells) are cultured in the presence of a CD131 binding compound and IL-3. Levels of tnfα secretion are then assessed by, for example, ELISA.

In further embodiments, the CD131 binding compounds of the present disclosure reduce or prevent IL-3 induced IL-13 release from human mast cells. For example, human cultured mast cells (e.g., ten week old peripheral blood derived cells) are cultured in the presence of a CD131 binding compound and IL-3. The level of IL-13 secretion is then assessed by, for example, ELISA.

In further embodiments, CD131 binding compounds of the present disclosure reduce or prevent IgE-mediated enhancement of IL-8 release from human mast cells by IL-3 and/or IL-5 and/or GM-CSF. For example, human cultured mast cells (e.g., ten weeks old peripheral blood derived cells) are cultured in the presence of a CD131 binding compound and IL-3/IL-5/GM-CSF (e.g., for about 48 hours). The cells are then incubated with IgE (e.g., human myeloma IgE) for a suitable period of time (e.g., about 24 hours), and IL-8 secretion is assessed, for example, by ELISA.

In further embodiments, the CD131 binding compound reduces or prevents the formation of CFU-GM from CD34+ human bone marrow cells (or cord blood cells) cultured in the presence of SCF, GM-CSF, IL-3, and IL-5. For example, CD34+ cells (e.g., about 1X 10 ³ Individual cells) are cultured in the presence of a CD131 binding compound (e.g., on methylcellulose (e.g., 1% methylcellulose) supplemented with fetal bovine serum, bovine serum albumin, SCF, GM-CSF, IL-3, and IL-5). The cells are cultured for a suitable time (e.g., about 16 days) and then the number of colonies formed is counted.

In further embodiments, the CD131 binding compound reduces survival or induces death of immune cells (e.g., eosinophils) from sputum or nasal polyp tissue of a subject suffering from an inflammatory airway disease or nasal polyp. For example, immune cells are cultured in the presence of IL-3 and/or IL-5 and/or GM-CSF, and proteins or antibodies. Cell death is then assessed using standard methods, for example by detecting expression of annexin-V (e.g., using fluorescence activated cell sorting).

In another embodiment, the CD131 binding compound reduces or prevents IL-3 mediated histamine release from basophils. For example, low density leukocytes comprising basophils are incubated with IgE, IL-3, and various concentrations of antibodies or antigen binding fragments. Control cells did not contain immunoglobulin (positive control) or IL-3 (negative control). Standard techniques (e.g., RIA) are then used to assess the level of histamine released. CD131 binding compounds that reduce the level of histamine release to a level below that of the positive control are believed to neutralize IL-3 signaling. In one embodiment, the reduced level is related to protein concentration. Exemplary methods for assessing IL-3 mediated histamine release are described, for example, in Lopez et al, J.Cell.Physiol.), 145:69, 1990.

Another assay for assessing IL-3 signaling neutralization involves determining whether a CD131 binding compound reduces or prevents IL-3-mediated effects on endothelial cells. For example, human Umbilical Vein Endothelial Cells (HUVECs) are cultured in the presence of IL-3 (optionally together with IFN-gamma) and various concentrations of CD131 binding compounds. The amount of secreted IL-6 is then assessed, for example, using an enzyme-linked immunosorbent assay (ELISA). Control cultures did not contain CD131 binding compounds (positive control) or IL-3 (negative control). CD131 binding compounds that reduce or prevent the production of IL-6 in the presence of IL-3 to levels below that of the positive control are believed to neutralize IL-3 signaling.

The present disclosure contemplates other methods for assessing neutralization of GM-CSF, IL-5, or IL-3 signaling.

Determining effector function

As described herein, some CD131 binding compounds have reduced effector function or have effector function (or enhanced effector function). Methods for assessing ADCC activity are known in the art.

In one embodiment, use is made of ⁵¹ Cr release assay, europium release assay or ³⁵ S release assay to assess the level of ADCC activity. In each of these assays, cells expressing CD131 are incubated with one or more of the compounds for a period of time and under conditions sufficient for the compounds to be taken up by the cells. At the position of ³⁵ In the case of the S release assay, cells expressing CD131 can be isolated from ³⁵ S-labeled methionine and/or cysteine are incubated together for a time sufficient for the labeled amino acids to be incorporated into the newly synthesized protein. The cells are then cultured in the presence or absence of a CD131 binding compound and in the presence of immune effector cells (e.g., peripheral Blood Mononuclear Cells (PBMCs) and/or NK cells). Then detecting the cell culture medium ⁵¹ Cr, europium and/or ³⁵ The amount of S and little or no change in the presence of the CD131 binding compound as compared to the absence of the CD131 binding compound indicates that the protein has a reduced effector function and an increased amount (or an increased amount as compared to the presence of the CD131 binding compound comprising an IgG1 Fc region) as compared to the absence of the CD131 binding compound, indicating effector function or enhanced effector function. Exemplary publications disclosing assays for assessing the level of ADCC induced by proteins include Hellstrom et al, proc. Natl. Acad.Sci. USA 83:7059-7063, 1986 and Bruggemann et al, journal of laboratory medicine (j.exp.med.) 166:1351-1361, 1987.

Other assays for assessing the level of ADCC induced by a protein include ACTI for flow cytometry ^TM Nonradioactive cytotoxicity assays (Celltechnology Co., california, USA) or CytotoxNonradioactive cytotoxicity assay (Promega, wisconsin, usa).

A C1q binding assay may also be performed to confirm that CD131 binding compounds are capable of binding C1q and may induce CDC. For evaluation of complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al J.Immunol. Methods) 202:163, 1996.

Determination of half-life

Some proteins encompassed by the present disclosure have improved half-lives, e.g., are modified to extend their half-lives as compared to unmodified proteins. Methods for determining proteins with improved half-lives will be apparent to the skilled artisan.

The half-life of the proteins of the present disclosure may be measured by in vivo pharmacokinetic studies, e.g., according to Kim et al, journal of immunology (Eur J of Immunol), 24:542 The method described in 1994. According to this method, radiolabeled protein is intravenously injected into mice and its plasma concentration is measured periodically as a function of time, for example 3 minutes to 72 hours after injection. Alternatively or additionally, other species, such as cynomolgus monkey and human, may be used, and/or non-radiolabeled proteins may be injected followed by detection using an enzyme-linked immunosorbent assay (ELISA). The clearance curve thus obtained should be biphasic, i.e. alpha phase and beta phase. To determine the in vivo half-life of the protein, clearance of the β phase was calculated and compared to either the wild-type protein or the unmodified protein.

The relative affinity of a protein to bind to a neonatal Fc receptor (FcRn) may also be indicative of its relative in vivo half-life (see, e.g., kim et al, J.European immunol.), 24:2429, 1994.

Therapeutic efficacy

Therapeutic efficacy of a compound that binds to CD131 can be assessed by comparing the severity of the disease or symptom in a subject administered the compound relative to a subject not administered the compound. Alternatively or additionally, the therapeutic efficacy of the candidate compound may be assessed in an animal model.

Intratracheal Lipopolysaccharide (LPS) -induced pulmonary inflammation is a well-known and well-documented animal model of ARDS (see, e.g., matute-Bello et al, 2011, journal of respiratory system cells and molecular biology (am. J. Respir. Cell mol. Biol.), 44, 725-738; orfanos et al, 2004, intensive Care medicine (Intensive Care Med.), 30, 1702-1714; tshima et al, 2009, medical science (International. Med.), 48, 621-630).

In an LPS-induced ARDS animal model, the efficacy of a candidate compound can be assessed by measuring the extent of inflammation in the lungs relative to a suitable control (i.e. placebo) animal. Inflammation in the lung can be assessed by measuring cell counts from bronchoalveolar lavage (BAL), and levels of total protein or pro-inflammatory cytokines in BALF and lung parenchyma homogenates. LPS-induced permeability (i.e., the extent of acute lung injury) in the lungs can also be measured.

Influenza A Virus (IAV) lung infection in C57BL/6 mice can reproduce to a large extent the immunopathological features of SARS-COV-2 infection in hACE2 transgenic mice (see, e.g., winkler ES et al 2020, nat immunology, 21 (11): 1327-1335).

In one embodiment, treatment efficacy can be assessed in an animal model of IAV-induced viral pneumonia. For example, IAV models that lead to excessive inflammation and viral pneumonia. In the IAV viral pneumonia model, the efficacy of candidate compounds can be assessed by measuring the extent of inflammation in the lungs of animals relative to a suitable control (i.e., placebo). Inflammation in the lung can be assessed by measuring cell counts from BAL and levels of total protein or pro-inflammatory cytokines in BALF and lung parenchyma homogenates.

In some embodiments, assessing the therapeutic efficacy of a compound comprises detecting and/or quantifying the expression level of a biomarker in a subject. Suitable biomarkers for assessing the efficacy of treatment of ARDS include G-CSF, plasminogen activator inhibitor-1 (PAI-1), D-dimer, neutrophil elastase, soluble AGE receptor (sRAGE), interferon gamma (IFN- γ), interleukin 1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and tumor necrosis factor alpha (TNF- α).

Detection and/or quantification of the biomarker may be performed by any method known in the art. For example, in one embodiment, the level of a biomarker is assessed using mass spectrometry. Mass spectrometry can be performed in combination with, for example, ultra-high performance liquid chromatography (UPLC), high Performance Liquid Chromatography (HPLC), gas Chromatography (GC), gas chromatography/mass spectrometry (GC/MS), and UPLC. Other methods of assessing the level of a biomarker include biological methods such as, but not limited to, ELISA assays, western blots, multiplex immunoassays, and the like. Other techniques may include the use of quantitative arrays, PCR, northern blot analysis. In order to determine the levels of a component or factor, it is not necessary to have an intact component (e.g., a full-length protein or an intact RNA transcript) present or to completely sequence it. In other words, determining, for example, the level of a fragment of the protein being analyzed may be sufficient to infer or evaluate whether the level of the biomarker being analyzed is increased or decreased. Similarly, if component levels are determined, for example, using an array or blot, the presence/absence/intensity of a detectable signal may be sufficient to assess the level of a biomarker.

To assess the level of a biomarker, a sample may be taken from a subject. The sample may or may not be processed prior to determining the level of the components of the biomarker profile. For example, whole blood may be obtained from an individual, and a blood sample may be processed (e.g., centrifuged) to separate plasma or serum from the blood. The sample may or may not be stored, such as frozen, prior to processing or analysis.

Biological samples that may be tested in the methods of the invention include whole blood, serum, plasma, tracheal aspirate, BALF, urine, saliva or other bodily fluids (stool, tears, synovial fluid, sputum), respiration (e.g., condensed respiration), or extracts or purifications thereof, or dilutions thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from living subjects or post-mortem. Samples may be prepared, for example diluted or concentrated where appropriate, and stored in a conventional manner.

Composition and method for producing the same

In some embodiments, a CD131 binding compound as described herein can be administered orally, parenterally, by inhalation spray, by adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implanted reservoir in a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, intraperitoneal and intracranial injection or infusion techniques.

Methods for preparing CD131 binding compounds into suitable forms (e.g., pharmaceutical compositions) for administration to a subject are known in the art and include, for example, as described in the Remington's Pharmaceutical Sciences (18 th edition, mike Publishing co.), easton, pa, 1990) and U.S. pharmacopoeia: methods described in the national formulary (U.S. Pharmacopeia: national Formulary) (mike publishing company, iston, pa., 1984).

The pharmaceutical compositions of the present disclosure are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ or joint. The composition for administration will typically comprise a solution of the CD131 binding compound dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers can be used, such as buffered saline and the like. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the CD131 binding compounds of the present disclosure in these formulations can vary widely and will be selected based primarily on liquid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles (vehicle) such as mixed oils and ethyl oleate may also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

After formulation, the CD131 binding compounds of the present disclosure will be administered in a manner compatible with the dosage formulation and in a therapeutically/prophylactically effective amount. Formulations are readily administered in a variety of dosage forms, such as injectable solutions of the type described above, but other pharmaceutically acceptable forms are also contemplated, such as tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms, and the like. Pharmaceutical "slow release" capsules or compositions may also be used. Sustained release formulations are typically designed to provide a constant drug level over an extended period of time and may be used to deliver the CD131 binding compounds of the present disclosure.

WO2002/080967 describes compositions and nebulized compositions and methods for administration comprising antibodies for the treatment of respiratory conditions, which are also suitable for administration of compounds according to the methods of the present disclosure.

Combination therapy

In one embodiment, the compounds of the present disclosure are administered in combination with another therapy useful for treating or preventing ARDS, either as a combination or additional therapeutic step, or as an additional component of a therapeutic formulation.

In some embodiments, the other therapy is a therapy commonly used to treat or prevent ARDS. Contemplated therapies for ARDS in combination with the methods of the present disclosure include treatments involving reducing pulmonary inflammation, reducing oedema, reducing alveolar and/or endothelial inflammation, treating the underlying cause of ARDS, and/or alleviating another symptom of ARDS. Another therapy may include administration of a compound, cell, or other molecule, and/or another therapy may include a physical or mechanical form of therapy, such as artificial ventilation and prone position.

In some embodiments, another therapy includes prone position, fluid management, oxygenation, artificial ventilation (including newer mechanical ventilation modes including but not limited to high frequency oscillatory ventilation), glucocorticoids, surfactants, inhaled nitric oxide, antioxidants, protease inhibitors, recombinant human activated protein C, β2-agonists, risophyllin, statins, inhaled heparin, diuretics, sedatives, analgesics, muscle relaxants, antivirals, antibiotics, inhaled prostacyclin, inhaled synthetic prostacyclin analogs, ketoconazole, alprostadil, keratinocyte growth factor, β -agonists, human mabs against TS factor 7a, interferon receptor agonists, insulin, perfluorocarbons, budesonide, recombinant human ACE, recombinant human CC10 protein, tissue plasminogen activator, human mesenchymal stem cells, or nutritional therapies. In other examples of combination therapies, another therapy is a glucocorticoid such as, for example, methylprednisolone, dexamethasone, prednisone, prednisolone, betamethasone, triamcinolone, budesonide (triamcinolone acetonide budesonide), and beclomethasone; beta-agonists such as, for example, albuterol; risoprotein (lisofyline); rosuvastatin (rosuvastatin), inhaled heparin; inhalation type nitric oxide; recombinant human activated protein C; NSAIDS such as, for example, ibuprofen (ibuprofen); naproxen (naproxen) and acetaminophen (acetaminophen); cis atracurium besylate (cisatracurium besylate); propylcysteine (procaysteine); acetylcysteine (acetylcysteine); inhaled prostacyclin; ketoconazole; alprostadil; keratinocyte growth factor; human mAb against TS factor 7 a; insulin; perfluorocarbon, recombinant human ACE; recombinant human CC10 protein; tissue plasminogen activator; human mesenchymal stem cells; or nutritional therapies such as the combination of omega-3 fatty acids, antioxidants and gamma-linolenic acid with isocaloric foods and external membrane oxygenation (ECMO).

NSAIDS includes, but is not limited to, aspirin, acetaminophen, diflunisal, bissalicylate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin (oxaprozin), loxoprofen (loxoprofen), indomethacin (indomethacin), tolmetin (tolmetin), sulindac (sulindac), etodolac (etodolac), ketorolac (ketoloac), nabumetone (nabumetone), diclofenac (dichlofenac), piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenaciram), droxicam (droxyac), lornoxicam (1 omimetm), isoxicam (isoxafenamic), mefenamic acid (meclofenamic acid), meclofenamic acid (meclofenamic acid), flufenamic acid (etofenamic acid), etofenamic acid (35) and etofenamic acid (etofenamic acid).

Analgesics include, but are not limited to, NSAIDS and opioids (anesthetics). Opioids include, but are not limited to, dexropropoxy-phen (dexropropoxy), codeine (codeine), tramadol (tramadol), tapentadol (tapentadol), anilididine (anilidine), alfa-odine (alphaprodine), pethidine (pethidine), hydrocodone (hydrocodone), morphine (morphine), oxycodone (oxycodone), methadone (methadone), diacetyl morphine (diamorphine), hydromorphone (hydromorphone), oxymorphone (oxymorphone), levorphanol (levorphanol), 7-hydroxy morphine, buprenorphine (buprenorphine), fentanyl (fentanil), sufentanil (sufentanil), bromocriptine (bromomadine), etorphine (etorphine), dihydroetorphine (dihydroorphine), and fentanyl (tetracaine).

Glucocorticoids include, but are not limited to, hydrocortisone (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone acetonide, beclomethasone, or fludrocortisone.

In some embodiments, the other therapy is a standard of care therapy. Standard of care therapies commonly used to treat or prevent ARDS include treatment of underlying conditions (e.g., infection), mechanical or non-invasive ventilation, fluid and hemodynamic therapy, prone position, treatment of opportunistic infections, nutritional and pharmacological therapies. For example, the intensive care medical institute (FICM) and the Intensive Care Society (ICS) have recently issued guidelines for standard care therapy for adult patients with ARDS (Griffiths et al, 2019, open acquisition journal of BMJ respiratory disease research (BMJ Open Resp Res) 6:e000420). In the case of mechanical ventilation, it is recommended to use low tidal volumes (< 6ml/kg ideal body weight) and airway pressures (plateau pressure < 30cmH 2O). For patients with moderate/severe ARDS (PaO ₂ /FiO ₂ Ratios less than or equal to 150 mmHg), a prone position of at least 12 hours per day is recommended. For all patients, the use of conservative fluid tubing is recommended The physical strategy, but for arterial oxygen partial pressure and inhaled oxygen fraction (PaO) ₂ /FiO ₂ ) Patients with ARDS with ratios less than or equal to 200mmHg and 150mmHg, respectively, were advised to mechanically ventilate with high positive end-expiratory pressure and to use the neuromuscular blocker cis-atracurium for 48 hours. The adventitial oxygen is suggested as an adjunct to protective mechanical ventilation in patients with very severe ARDS. The methods of the present disclosure may be performed in combination with any of the above therapies for treating or preventing ARDS.

In some embodiments, the other therapy is a therapy for treating the root cause of ARDS. For example, in one embodiment, another therapy includes administration of an antiviral agent or antibiotic (e.g., where the root cause of ARDS is an infection). In one embodiment, another therapy comprises administration of adefovir.

In one embodiment, the compound that binds to CD131 is administered concurrently with another therapy. In one embodiment, the compound that binds to CD131 is administered prior to another therapy. In one embodiment, the compound that binds to CD131 is administered after another therapy.

In some embodiments, the compound that binds to CD131 is administered in combination with a cell. In some embodiments, the cell is a stem cell, such as a mesenchymal stem cell. In some embodiments, the compound that binds to CD131 is administered in combination with gene therapy.

Dosage and time of administration

The appropriate dosage of the CD131 binding compounds of the present disclosure will vary depending on the particular CD131 binding compound, the condition to be treated, and/or the subject being treated. It is within the ability of skilled practitioners to determine the appropriate dosage, for example, by starting with a sub-optimal dosage and incrementally modifying the dosage to determine the optimal or useful dosage. Alternatively, to determine the appropriate dose for treatment/prophylaxis, data from cell culture assays or animal studies are used, where the appropriate dose is within a range of circulating concentrations including ED of the active compound with little or no toxicity ₅₀ . The dosage may depend on the dosage form employed and the dosage form utilizedThe route of administration varies within this range. The therapeutically or prophylactically effective dose can be estimated initially from cell culture assays. The dose may be formulated in animal models to achieve a composition comprising IC as determined in cell culture ₅₀ (i.e., the concentration or amount of compound that achieves half-maximal inhibition of symptoms (half-maximal inhibition)). Such information can be used to more accurately determine an effective dose in humans. The level in the plasma may be measured, for example, by high performance liquid chromatography.

In some embodiments, the methods of the present disclosure comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the prognosis and/or status of the subject and/or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed and accepted as a clinical diagnosis or clinical feature of the condition. The amount to be administered to a subject will depend on the specific characteristics of the condition to be treated, the type and stage of the condition to be treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and weight. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. Thus, this term should not be construed to limit the disclosure to a particular amount, e.g., weight or amount of protein, but rather the disclosure encompasses any amount of CD131 binding compound sufficient to achieve the result in a subject.

As used herein, the term "prophylactically effective amount" should be understood to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. Those of skill in the art will appreciate that such amounts will vary depending, for example, on the particular C131 binding protein administered and/or the type or severity or level of the particular subject and/or condition and/or the predisposition to (genetic or otherwise) the condition. Thus, this term should not be construed to limit the disclosure to a particular amount, e.g., weight or amount of CD131 binding compound, but the disclosure encompasses any amount of C131 binding protein sufficient to achieve the result in a subject.

For in vivo administration of the CD131 binding compounds described herein, the normal dose may vary from about 10ng/kg of body weight of the subject to about 100mg/kg of body weight of the subject or more per day. For repeated administration over several days or longer, depending on the severity of the disease or condition to be treated, the treatment may continue until the desired symptom suppression is achieved.

In some embodiments, the CD131 binding compound is administered at an initial (or loading) dose of between about 1mg/kg to about 30mg/kg, such as about 1mg/kg to about 10mg/kg, or about 1mg/kg or about 2mg/kg or 5mg/kg. The CD131 binding compound may then be administered at a lower maintenance dose of between about 0.01mg/kg to about 2mg/kg, such as about 0.05mg/kg to about 1mg/kg, such as about 0.1mg/kg or 0.5mg/kg or 1mg/kg. The maintenance dose may be administered every 7-30 days, such as every 10-15 days, for example every 10 or 11 or 12 or 13 or 14 or 15 days.

In some embodiments, the CD131 binding compound is administered at a dose of between about 0.01mg/kg and about 50mg/kg, such as between about 0.05mg/kg and about 30mg/kg, such as between about 0.1mg/kg and about 20mg/kg, such as between about 0.1mg/kg and about 10mg/kg, such as between about 0.1mg/kg and about 2 mg/kg. For example, the CD131 binding compound is administered at a dose of between about 0.01mg/kg and about 5mg/kg, such as about 0.1mg/kg to about 2mg/kg, such as about 0.2mg/kg or 0.3mg/kg or 0.5mg/kg or 1mg/kg or 1.5mg/kg (e.g., without a higher loading dose or a lower maintenance dose). In some embodiments, for example, a large number of doses are administered every 7-30 days, such as every 10-22 days, for example every 10-15 days, for example every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, the CD131 binding compound is administered every 7 days or every 14 days or every 21 days.

In some embodiments, the mammal is administered the CD131 binding compound for no more than 7 consecutive days or 6 consecutive days or 5 consecutive days or 4 consecutive days at the beginning of the therapy.

In the case of a mammal that does not respond adequately to treatment, multiple doses may be administered within a week. Alternatively or additionally, an increased dose may be administered.

In another embodiment, for mammals experiencing adverse effects, the initial (or loading) doses may be separated over a number of days in a week or over a number of consecutive days.

According to the methods of the present disclosure, administration of the CD131 binding compound may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled artisan. Administration of the CD131 binding compound may be substantially continuous over a preselected period of time, or may be a series of spaced doses, for example during or after the development of a condition.

Kit for detecting a substance in a sample

Another embodiment of the present disclosure provides a kit containing a compound for treating or preventing ARDS as described above.

In one embodiment, the kit comprises: (a) A container comprising a compound that binds to CD131 as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert having instructions for treating, preventing, or alleviating the effects of ARDS in a subject.

According to this embodiment of the present disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of various materials such as glass or plastic. The container holds or holds a composition effective to treat or prevent ARDS and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a compound that binds to CD 131. The label or package insert indicates that the composition is to be administered to a subject suitable for treatment, e.g., a subject suffering from or at risk of developing ARDS, wherein specific instructions are provided regarding the amount and interval of administration of the compound and any other drug. The kit may further comprise additional containers comprising pharmaceutically acceptable dilution buffers, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and/or dextrose solution. The kit may further comprise other materials, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user perspective.

The present disclosure includes the following non-limiting examples.

Examples

Example 1: anti-CD 131/βc and βIL3 chimeric monoclonal antibodies reduce neutrophil and macrophage accumulation in the lung

Antibodies to

A "chimeric mAb" is a chimeric mouse monoclonal antibody comprising a rat Fab region which recognizes the extracellular regions of the mouse CD131/βc and βIL3 cytokine receptor subunits and inhibits signaling through these receptors and mouse IgG1 Fc. Isotype control antibodies were used for comparison (muBM 4-muGIK RP3204 or "BM 4").

Acute Respiratory Distress Syndrome (ARDS) model

Wild-type mice (8-12 week old c57BL/6 mice) were used. Mouse colonies were obtained from animal resource centers (Animal Resources Centre) (ARC) (Canning Vale, western Australia, australia (Western Australia, australia)). Female mice eight to twelve weeks old were used for experiments conducted according to ethical guidelines of the national health and medical research committee (National Health and Medical Research Council ofAustralia) in australia.

ARDS model: mice were anesthetized and intubated into the mouth and trachea with a cannula attached to a 50ml Hamilton syringe by intratracheal (i.t.) installation of 3 μg of escherichia coli LPS (LPS; 0111: b4, sigma-Aldrich) into the lung. Mice were terminated 24 hours after LPS cannulation by deadly injection of pentobarbital.

Evaluation of ARDS model: to evaluate the effect of prophylactic and therapeutic administration of chimeric mAb in ARDS model, mice were intravenously (i.v.) administered 50mg/kg of chimeric mAb or BM4 before (prophylactic) 24 hours or after (therapeutic) 6 hours of LPS cannulation. Bronchoalveolar lavage fluid (BALF) was obtained by catheterizing the trachea with a 20G catheter. Two lungs were lavaged three times (0.4 ml PBS per aliquot) with an average BALF return of 0.9-1.1 ml/mouse.

Cell assay for BALF

The total cell number in BALF was determined by counting with a cytometer. Differential counts were performed by cell centrifuged preparations (cytosine 4;Thermo Scientific), fixed and stained with Geimsa (Sigma). Differential counts were based on a count of 200 cells, which were classified as neutrophils, macrophages or lymphocytes using standard morphological criteria. The counts were performed by individual observers blinded to the treatment group.

A significant reduction in cell numbers in BALF was observed in ARDS models with prophylactic or therapeutic administration of chimeric mAb compared to BM4 group (fig. 1A and 2A). This decrease was associated with the decrease in macrophages and neutrophils observed in BALF following prophylactic or therapeutic administration of the chimeric mAb (fig. 1B and 2B).

Example 2: chimeric monoclonal antibodies against CD131/βc and βIL3 inhibit GM-CSF and IL-3 induced proliferation of FDCP1 cells

FDCP1 mouse bone marrow-derived cell lines were made deficient in 10% Fetal Calf Serum (FCS) for 3h and were treated at 1X 10 in the dose response of isotype control monoclonal antibody (isotype control mAb) or chimeric monoclonal antibody (chimeric mAb) as used in example 1 ⁴ The density of individual cells/wells was plated in 200 μl RPMI/10% fcs. ED was added to each well after 1 hour of pre-incubation with isotype control mAb or chimeric mAb ₅₀ Doses of mIL3 (0.54 ng/ml) or mGM-CSF (0.022 ng/ml).

Proliferation was measured after 48 hours incubation with either mIL3 or mGM-CSF. FDCP1 cells at 37 ℃/5% CO ₂ Incubations were continued for 1h in 20. Mu.l/well Cell Titre96 Aqueous One Solution (Promega). Optical Density (OD) readings were made at 490nm to 690nm and data were entered into Prism.

FIG. 3 shows that chimeric mAbs reduce proliferation of FDCP1 cells by inhibiting GM-CSF and IL-3 signaling in a dose-dependent manner.

Example 3: in the hβ cTg mouse model of Acute Respiratory Distress Syndrome (ARDS), CSL311 reduces cellular inflammation, reduces histopathology and increases blood oxygenation

Antibodies to

CSL311 (ahu A2-G4pK-VR 24-29) is a human antibody that targets the common cytokine binding site (site 2) of human βc (CD 131) homodimers. In SEQ ID NO:14, and in SEQ ID NO:15 provides the light chain amino acid sequence. Control antibodies of the human IgG4 (chBM 4-G4 pK) isotype were used for comparison.

Acute Respiratory Distress Syndrome (ARDS) model

To determine the therapeutic potential of CSL311 for the treatment of ARDS, markers of lung leukocyte numbers and lung injury were examined in a transgenic mouse model expressing human βc receptor. Transgenic mice, termed "hβ cTg", are transgenic for (i) the human βc receptor and (ii) two endogenous β subunits (βc ^-/- /β _IL-3 ^-/- ) Is homozygous for the knockout of (c). ARDS was induced in hβ cTg mice by nasal instillation of 10 μg of E.coli lipopolysaccharide (LPS; 026:B6, sigma-Aldrich) for 24 hours. Mice were treated intravenously with CSL311 (50 mg/kg) or isotype control mAb 3h prior to LPS challenge.

Direct lung instillation of LPS resulted in significant weight loss in the hβ cTg mice challenged with LPS, which was significantly reduced with CSL311 administration (fig. 4A). LPS challenge significantly increased the number of blood neutrophils and monocytes in hβ cTg mice, which was reduced to control levels with CSL311 administration (fig. 4B and 4C). Neutrophils migrate into the bronchoalveolar lavage (BAL) airway compartment in response to LPS challenge, which is significantly reduced by about 70% with CSL311 administration (fig. 4D). Histological scoring of lung inflammation showed that CSL311 significantly reduced lung injury caused by acute LPS challenge in h beta cTg mice (fig. 4E and 4F). Peak lung Myeloperoxidase (MPO) levels were also significantly reduced, consistent with a significant reduction in leukocyte infiltration of lung tissue (fig. 4G). Furthermore, CSL311 significantly reduced peak edema caused by LPS challenge, as assessed by quantifying total protein levels of BALF (fig. 4H).

In the case of acute LPS challenge to hβ cTg mice, release of Neutrophil Extracellular Trap (NET) associated with NETopathic tissue damage in response to acute injury or infection was observed (fig. 5A). FIG. 5A shows staining of lung tissue sections of LPS-challenged hβ cTg mice with antibodies against MPO and citrullinated histone H3. In hβ cTg mice, NET formation was significantly increased at higher doses of LPS challenge (10 μg) (fig. 5A). NET was also quantified in BALF of LPS challenged hβ cTg mice, showing peak MPO and dsDNA levels (10 g) 24 hours after LPS challenge (fig. 5B and 5C). Peak MPO and dsDNA levels were significantly reduced with CSL311 administration prior to LPS challenge in hβ cTg mice (fig. 5D and 5E).

To determine if CSL311 helped maintain pulmonary function in response to ARDS, blood oxygenation was examined in hβ cTg mice. ARDS was induced in hβ cTg mice by intranasal instillation of 20 μl of 5 μg/mL LPS. Mice were treated intravenously with CSL311 (50 mg/kg) or isotype control mAb (50 mg/kg) 30 minutes prior to LPS challenge. Mice treated with intranasal PBS were used to determine blood oxygenation in the absence of LPS challenge. Using Jr pulse oximeter @Jr.pulse oxidation) and heart rate monitor (Kent Scientific) monitor the percentage of blood oxygenation 16h, 48h and 72h after administration of LPS or PBS.

Administration of LPS to hβ cTg mice resulted in acute reduction of blood oxygenation, which peaked at about 16h and began to regress thereafter (fig. 6, LPS-ISO group). Administration of CSL311 significantly protected the hβ cTg mice from LPS-induced reduction in blood oxygenation at 16h, 48h and 72h compared to isotype control antibodies (fig. 6). Taken together, these data demonstrate the effectiveness of targeting the βc receptor (CD 131) for the treatment and prevention of ARDS.

Example 4: immunopathology and excessive inflammation in preclinical mouse models of CSL311 reduced viral pneumonia

Viral pneumonia model

Viral pneumonia was induced in transgenic hβ cTg mice. Briefly, under mild isoflurane anaesthesia, influenza a virus (IAV, HKx31, H3N2 strain, 10 ⁴ PFU) intranasal infection of hβ cTg mice.

In hβ cTg mice, expression of Gm-csf in lung tissue increased significantly during the early stages of IAV infection (day 3) and decreased on day 6 (fig. 7A). In contrast, the expression of Il3 and Il5 peaked at a later time point on day 6 (fig. 7B and 7C). Expression of the βc receptor transcript (CSF 2 RB) was significantly increased on days 3 and 6 post IAV infection, consistent with continuous tissue infiltration of inflammatory cells expressing the βc receptor during acute viral infection (fig. 7D).

Mice were treated with either single dose of CSL311 (50 mg/kg) or isotype control mAb (50 mg/kg) via intravenous injection 4 days after IAV infection. On day 6, IAV-infected mice showed a significant decrease in body weight, and this was not altered by CSL311 treatment (fig. 8A). Pulmonary viral load was determined by RTqPCR on viral polymerase a subunit (PA) gene and high levels of viral RNA were detected in IAV infected mice. The levels were the same in CSL311 treated mice (fig. 8B). Blocking of the βc receptor by treatment with CSL311 significantly reduced the blood granulocytosis and mononucleosis induced by IAV infection (fig. 8C and 8D). Furthermore, the elevation of blood Hemoglobin (HGB) levels, which may be secondary to IAV-induced hypoxia, was completely prevented by CSL311 treatment (fig. 8E). In IAV infected mice, the numbers of neutrophils (fig. 8F) and macrophages (fig. 8G) in the bronchoalveolar lavage (BAL) compartment were significantly increased, while CSL311 significantly reduced both myeloid cell populations. Furthermore, IAV infection caused significant bleeding of the entire lung lobes, as characterized by the appearance of dark purple colored lung lobes, as opposed to pink lung from saline-treated mice. CSL311 treatment is associated with a reduction in bleeding areas of the entire lung lobes.

To further characterize immunological changes in the lung following CSL311 treatment of IAV-infected hβ cTg mice, the immune response was measured by flowMyeloid cells (fig. 9A) and lymphoid cells (fig. 9G) were analyzed by cytometry. Briefly, after BAL, the upper leaves of the lungs were finely minced and digested in Liberase TM (Sigma, usa) at 37 ℃ with continuous shaking. Single cell suspensions were prepared by passing digested tissue through a 25G needle and then through a 40 μm cell filter. The lung cells were pelleted and the erythrocytes lysed with ACK lysis buffer. After blocking with CD16/CD32 antibody, the cells were blocked with a cell-free antibody containing FITC-CD45, PE-Siglec F, APC-F4/80, eFluor 450-CD11b, PE/Cy7-CD11c, perCp/eFluor710-Ly6G and LIVE/DEAD ^TM Fixable yellow DEAD cell stain (LIVE/DEAD) ^TM Fixab le Yellow Dead Cell Stain) or a mixture of myeloid cell antibodies comprising APC/eFluor 780-CD45, perCP/cyanine5.5-CD3e, PE/Cy7-NK-1.1 (BD, USA), PE-CD8a (BioLegend, USA), PE/eFluor 610-CD4, alexa Fluor 488-FOXP3 (BioLegend, USA) and LIVE/DEAD ^TM Cells were stained with a mixture of lymphoid cells antibodies that were fixable to a purple dead cell stain, as previously described 23, 26, 27.Foxp 3/transcription factor staining buffer was used for cell permeabilization prior to Foxp3 staining. After staining, cells were fixed with IC fixation buffer and then analyzed on BD FACSAria.

After CSL311 treatment, lung neutrophils (fig. 9B) and macrophages (fig. 9C-E) were significantly reduced in IAV infected mice, consistent with the changes observed in blood and BAL. The decrease in macrophage numbers was due to normalization of alveolar macrophages (AM, fig. 9C) and inhibition of monocyte-derived exudative macrophages (EM, fig. 9D) and blood monocytes (fig. 9E) recruited into the lung. It was previously reported that the number of lung eosinophils was increased in IAV-infected mice, also in IAV-infected hβ cTg mice, and antagonism of the βc receptor by CSL311 significantly reduced this response (fig. 9F). In contrast to myeloid cells, a subset of lymphocytes that provide important protection against viral infection do not respond directly to βc cytokines. On day 6 after IAV infection, significant expansion of Natural Killer (NK) cells (fig. 9H) and NKT cells (fig. 9I) was observed, which was not significantly altered by CSL311 treatment. Immunosuppressive regulatory T (Treg) cells were increased by IAV infection in a manner that was unaffected by CSL311 treatment (fig. 9J). On day 6 post IAV infection, lung CD4 and cd8+ T cells did not increase and CSL311 treatment did not further alter their corresponding numbers (fig. 9K and 9L).

At the molecular level, lung expression of the neutrophil chemokine Cxcl1 gene, the monocyte/macrophage chemokine Ccl2 gene, and the eosinophil chemokine Cc124 gene was significantly increased in IAV-infected mice (fig. 10A-C). CSL311 treatment did not reduce Cxcl1 gene expression, but significantly reduced the Ccl2 and Ccl24 gene expression. These reductions were consistent with lung macrophages being the primary cellular source of CCL2 and CCL24 during IAV infection, which was reduced by CSL311 treatment. In IAV infected mice, the levels of Cxcl10 and Cxcl1 gene expression were increased in a manner that was not altered by CSL311 treatment (fig. 10D). Cxcl1 and Cxcl 0 can be produced by a variety of cell types, including fibroblasts and endothelial cells, which are not targets for βc antagonism. The gene expression of inflammatory cytokine Il1 alpha was increased in the lungs of IAV-infected mice, and the βc receptor blockade completely normalized Il1 alpha gene levels (fig. 10E). IL-1R signaling may regulate the expression of neutrophil adhesion molecules, and reduction of IL-1 alpha may help reduce neutrophil inflammation in the lung. Il6 gene expression was also increased in IAV-infected mice, and its level was not decreased in CSL 311-treated mice (fig. 10F). Interferon constitutes the first line of defense against viral defenses and all three types of interferon are upregulated in IAV infected lungs (fig. 10G-I). Treatment with CSL311 did not alter the level of type II interferon (Ifng) gene expression, consistent with no alteration in cell sources (NK/NKT cells and cytotoxic T cells) with CSL311 treatment. IAV-induced gene expression of type I (Ifnb) and type III interferons (Ifnl 2/3) was also retained in CSL 311-treated mice (fig. 10G and I), indicating their cellular origin (plasmacytoid dendritic cells (pDC) and lung epithelium) were functional in the context of βc receptor antagonism.

Sequence listing

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Trp Glu Arg Ser Leu Ala Gly Ala Glu Glu Thr Ile Pro Leu Gln Thr

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Leu Arg Cys Tyr Asn Asp Tyr Thr Ser His Ile Thr Cys Arg Trp Ala

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Asp Thr Gln Asp Ala Gln Arg Leu Val Asn Val Thr Leu Ile Arg Arg

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Val Asn Glu Asp Leu Leu Glu Pro Val Ser Cys Asp Leu Ser Asp Asp

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Met Pro Trp Ser Ala Cys Pro His Pro Arg Cys Val Pro Arg Arg Cys

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Val Ile Pro Cys Gln Ser Phe Val Val Thr Asp Val Asp Tyr Phe Ser

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Phe Gln Pro Asp Arg Pro Leu Gly Thr Arg Leu Thr Val Thr Leu Thr

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Gln His Val Gln Pro Pro Glu Pro Arg Asp Leu Gln Ile Ser Thr Asp

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Gln Asp His Phe Leu Leu Thr Trp Ser Val Ala Leu Gly Ser Pro Gln

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Ser His Trp Leu Ser Pro Gly Asp Leu Glu Phe Glu Val Val Tyr Lys

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Arg Leu Gln Asp Ser Trp Glu Asp Ala Ala Ile Leu Leu Ser Asn Thr

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Ser Gln Ala Thr Leu Gly Pro Glu His Leu Met Pro Ser Ser Thr Tyr

195 200 205

Val Ala Arg Val Arg Thr Arg Leu Ala Pro Gly Ser Arg Leu Ser Gly

210 215 220

Arg Pro Ser Lys Trp Ser Pro Glu Val Cys Trp Asp Ser Gln Pro Gly

225 230 235 240

Asp Glu Ala Gln Pro Gln Asn Leu Glu Cys Phe Phe Asp Gly Ala Ala

245 250 255

Val Leu Ser Cys Ser Trp Glu Val Arg Lys Glu Val Ala Ser Ser Val

260 265 270

Ser Phe Gly Leu Phe Tyr Lys Pro Ser Pro Asp Ala Gly Glu Glu Glu

275 280 285

Cys Ser Pro Val Leu Arg Glu Gly Leu Gly Ser Leu His Thr Arg His

290 295 300

His Cys Gln Ile Pro Val Pro Asp Pro Ala Thr His Gly Gln Tyr Ile

305 310 315 320

Val Ser Val Gln Pro Arg Arg Ala Glu Lys His Ile Lys Ser Ser Val

325 330 335

Asn Ile Gln Met Ala Pro Pro Ser Leu Asn Val Thr Lys Asp Gly Asp

340 345 350

Ser Tyr Ser Leu Arg Trp Glu Thr Met Lys Met Arg Tyr Glu His Ile

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Asp His Thr Phe Glu Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp Lys

370 375 380

Asp Ser Lys Thr Glu Thr Leu Gln Asn Ala His Ser Met Ala Leu Pro

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Ala Leu Glu Pro Ser Thr Arg Tyr Trp Ala Arg Val Arg Val Arg Thr

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Ser Arg Thr Gly Tyr Asn Gly Ile Trp Ser Glu Trp Ser Glu Ala Arg

420 425 430

Ser Trp Asp Thr Glu Ser Val Leu Pro Met Trp Val Leu Ala Leu Ile

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Val Ile Phe Leu Thr Ile Ala Val Leu Leu Ala Leu Arg Phe Cys Gly

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Ile Tyr Gly Tyr Arg Leu Arg Arg Lys Trp Glu Glu Lys Ile Pro Asn

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Pro Ser Lys Ser His Leu Phe Gln Asn Gly Ser Ala Glu Leu Trp Pro

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Pro Gly Ser Met Ser Ala Phe Thr Ser Gly Ser Pro Pro His Gln Gly

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Pro Trp Gly Ser Arg Phe Pro Glu Leu Glu Gly Val Phe Pro Val Gly

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Phe Gly Asp Ser Glu Val Ser Pro Leu Thr Ile Glu Asp Pro Lys His

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Val Cys Asp Pro Pro Ser Gly Pro Asp Thr Thr Pro Ala Ala Ser Asp

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Leu Pro Thr Glu Gln Pro Pro Ser Pro Gln Pro Gly Pro Pro Ala Ala

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Ser His Thr Pro Glu Lys Gln Ala Ser Ser Phe Asp Phe Asn Gly Pro

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Tyr Leu Gly Pro Pro His Ser Arg Ser Leu Pro Asp Ile Leu Gly Gln

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Pro Glu Pro Pro Gln Glu Gly Gly Ser Gln Lys Ser Pro Pro Pro Gly

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Ser Leu Glu Tyr Leu Cys Leu Pro Ala Gly Gly Gln Val Gln Leu Val

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Pro Leu Ala Gln Ala Met Gly Pro Gly Gln Ala Val Glu Val Glu Arg

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Arg Pro Ser Gln Gly Ala Ala Gly Ser Pro Ser Leu Glu Ser Gly Gly

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Gly Pro Ala Pro Pro Ala Leu Gly Pro Arg Val Gly Gly Gln Asp Gln

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Lys Asp Ser Pro Val Ala Ile Pro Met Ser Ser Gly Asp Thr Glu Asp

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Pro Gly Val Ala Ser Gly Tyr Val Ser Ser Ala Asp Leu Val Phe Thr

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Pro Asn Ser Gly Ala Ser Ser Val Ser Leu Val Pro Ser Leu Gly Leu

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Pro Ser Asp Gln Thr Pro Ser Leu Cys Pro Gly Leu Ala Ser Gly Pro

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Pro Gly Ala Pro Gly Pro Val Lys Ser Gly Phe Glu Gly Tyr Val Glu

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Leu Pro Pro Ile Glu Gly Arg Ser Pro Arg Ser Pro Arg Asn Asn Pro

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Val Pro Pro Glu Ala Lys Ser Pro Val Leu Asn Pro Gly Glu Arg Pro

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Ala Asp Val Ser Pro Thr Ser Pro Gln Pro Glu Gly Leu Leu Val Leu

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Gln Gln Val Gly Asp Tyr Cys Phe Leu Pro Gly Leu Gly Pro Gly Pro

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Leu Ser Leu Arg Ser Lys Pro Ser Ser Pro Gly Pro Gly Pro Glu Ile

835 840 845

Lys Asn Leu Asp Gln Ala Phe Gln Val Lys Lys Pro Pro Gly Gln Ala

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Val Pro Gln Val Pro Val Ile Gln Leu Phe Lys Ala Leu Lys Gln Gln

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Asp Tyr Leu Ser Leu Pro Pro Trp Glu Val Asn Lys Pro Gly Glu Val

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Cys

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Met Val Leu Leu Trp Leu Thr Leu Leu Leu Ile Ala Leu Pro Cys Leu

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Leu Gln Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met

20 25 30

Lys Ala Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr

35 40 45

Asp Ile Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn

50 55 60

Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn

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Tyr Thr Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe

85 90 95

Pro Glu Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys

100 105 110

Trp Ile His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro

115 120 125

Gly Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn

130 135 140

Arg Arg Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly

145 150 155 160

Thr Arg Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly

165 170 175

Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly

180 185 190

Ile Pro Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu

195 200 205

Thr Pro Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met

210 215 220

His Trp Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu

225 230 235 240

Gln Ile Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp

245 250 255

Arg Thr Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile

260 265 270

Arg Ala Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro

275 280 285

Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp

290 295 300

Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys

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Val Phe Val Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro

325 330 335

Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Ser Phe Gln Asn Asp

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Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu

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Val Thr Glu Val Gln Val Val Gln Lys Thr

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Met Ala Arg Leu Gly Asn Cys Ser Leu Thr Trp Ala Ala Leu Ile Ile

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Leu Leu Leu Pro Gly Ser Leu Glu Glu Cys Gly His Ile Ser Val Ser

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Ala Pro Ile Val His Leu Gly Asp Pro Ile Thr Ala Ser Cys Ile Ile

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Lys Gln Asn Cys Ser His Leu Asp Pro Glu Pro Gln Ile Leu Trp Arg

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Leu Gly Ala Glu Leu Gln Pro Gly Gly Arg Gln Gln Arg Leu Ser Asp

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Gly Thr Gln Glu Ser Ile Ile Thr Leu Pro His Leu Asn His Thr Gln

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Ala Phe Leu Ser Cys Cys Leu Asn Trp Gly Asn Ser Leu Gln Ile Leu

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Asp Gln Val Glu Leu Arg Ala Gly Tyr Pro Pro Ala Ile Pro His Asn

115 120 125

Leu Ser Cys Leu Met Asn Leu Thr Thr Ser Ser Leu Ile Cys Gln Trp

130 135 140

Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Thr Leu Lys Ser

145 150 155 160

Phe Lys Ser Arg Gly Asn Cys Gln Thr Gln Gly Asp Ser Ile Leu Asp

165 170 175

Cys Val Pro Lys Asp Gly Gln Ser His Cys Cys Ile Pro Arg Lys His

180 185 190

Leu Leu Leu Tyr Gln Asn Met Gly Ile Trp Val Gln Ala Glu Asn Ala

195 200 205

Leu Gly Thr Ser Met Ser Pro Gln Leu Cys Leu Asp Pro Met Asp Val

210 215 220

Val Lys Leu Glu Pro Pro Met Leu Arg Thr Met Asp Pro Ser Pro Glu

225 230 235 240

Ala Ala Pro Pro Gln Ala Gly Cys Leu Gln Leu Cys Trp Glu Pro Trp

245 250 255

Gln Pro Gly Leu His Ile Asn Gln Lys Cys Glu Leu Arg His Lys Pro

260 265 270

Gln Arg Gly Glu Ala Ser Trp Ala Leu Val Gly Pro Leu Pro Leu Glu

275 280 285

Ala Leu Gln Tyr Glu Leu Cys Gly Leu Leu Pro Ala Thr Ala Tyr Thr

290 295 300

Leu Gln Ile Arg Cys Ile Arg Trp Pro Leu Pro Gly His Trp Ser Asp

305 310 315 320

Trp Ser Pro Ser Leu Glu Leu Arg Thr Thr Glu Arg Ala Pro Thr Val

325 330 335

Arg Leu Asp Thr Trp Trp Arg Gln Arg Gln Leu Asp Pro Arg Thr Val

340 345 350

Gln Leu Phe Trp Lys Pro Val Pro Leu Glu Glu Asp Ser Gly Arg Ile

355 360 365

Gln Gly Tyr Val Val Ser Trp Arg Pro Ser Gly Gln Ala Gly Ala Ile

370 375 380

Leu Pro Leu Cys Asn Thr Thr Glu Leu Ser Cys Thr Phe His Leu Pro

385 390 395 400

Ser Glu Ala Gln Glu Val Ala Leu Val Ala Tyr Asn Ser Ala Gly Thr

405 410 415

Ser Arg Pro Thr Pro Val Val Phe Ser Glu Ser Arg Gly Pro Ala Leu

420 425 430

Thr Arg Leu His Ala Met Ala Arg Asp Pro His Ser Leu Trp Val Gly

435 440 445

Trp Glu Pro Pro Asn Pro Trp Pro Gln Gly Tyr Val Ile Glu Trp Gly

450 455 460

Leu Gly Pro Pro Ser Ala Ser Asn Ser Asn Lys Thr Trp Arg Met Glu

465 470 475 480

Gln Asn Gly Arg Ala Thr Gly Phe Leu Leu Lys Glu Asn Ile Arg Pro

485 490 495

Phe Gln Leu Tyr Glu Ile Ile Val Thr Pro Leu Tyr Gln Asp Thr Met

500 505 510

Gly Pro Ser Gln His Val Tyr Ala Tyr Ser Gln Glu Met Ala Pro Ser

515 520 525

His Ala Pro Glu Leu His Leu Lys His Ile Gly Lys Thr Trp Ala Gln

530 535 540

Leu Glu Trp Val Pro Glu Pro Pro Glu Leu Gly Lys Ser Pro Leu Thr

545 550 555 560

His Tyr Thr Ile Phe Trp Thr Asn Ala Gln Asn Gln Ser Phe Ser Ala

565 570 575

Ile Leu Asn Ala Ser Ser Arg Gly Phe Val Leu His Gly Leu Glu Pro

580 585 590

Ala Ser Leu Tyr His Ile His Leu Met Ala Ala Ser Gln Ala Gly Ala

595 600 605

Thr Asn Ser Thr Val Leu Thr Leu Met Thr Leu Thr Pro Glu Gly Ser

610 615 620

Glu Leu His Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Thr

625 630 635 640

Cys Leu Cys Gly Thr Ala Trp Leu Cys Cys Ser Pro Asn Arg Lys Asn

645 650 655

Pro Leu Trp Pro Ser Val Pro Asp Pro Ala His Ser Ser Leu Gly Ser

660 665 670

Trp Val Pro Thr Ile Met Glu Glu Asp Ala Phe Gln Leu Pro Gly Leu

675 680 685

Gly Thr Pro Pro Ile Thr Lys Leu Thr Val Leu Glu Glu Asp Glu Lys

690 695 700

Lys Pro Val Pro Trp Glu Ser His Asn Ser Ser Glu Thr Cys Gly Leu

705 710 715 720

Pro Thr Leu Val Gln Thr Tyr Val Leu Gln Gly Asp Pro Arg Ala Val

725 730 735

Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr Ser Asp Gln Val Leu Tyr

740 745 750

Gly Gln Leu Leu Gly Ser Pro Thr Ser Pro Gly Pro Gly His Tyr Leu

755 760 765

Arg Cys Asp Ser Thr Gln Pro Leu Leu Ala Gly Leu Thr Pro Ser Pro

770 775 780

Lys Ser Tyr Glu Asn Leu Trp Phe Gln Ala Ser Pro Leu Gly Thr Leu

785 790 795 800

Val Thr Pro Ala Pro Ser Gln Glu Asp Asp Cys Val Phe Gly Pro Leu

805 810 815

Leu Asn Phe Pro Leu Leu Gln Gly Ile Arg Val His Gly Met Glu Ala

820 825 830

Leu Gly Ser Phe

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Met Ile Ile Val Ala His Val Leu Leu Ile Leu Leu Gly Ala Thr Glu

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Ile Leu Gln Ala Asp Leu Leu Pro Asp Glu Lys Ile Ser Leu Leu Pro

20 25 30

Pro Val Asn Phe Thr Ile Lys Val Thr Gly Leu Ala Gln Val Leu Leu

35 40 45

Gln Trp Lys Pro Asn Pro Asp Gln Glu Gln Arg Asn Val Asn Leu Glu

50 55 60

Tyr Gln Val Lys Ile Asn Ala Pro Lys Glu Asp Asp Tyr Glu Thr Arg

65 70 75 80

Ile Thr Glu Ser Lys Cys Val Thr Ile Leu His Lys Gly Phe Ser Ala

85 90 95

Ser Val Arg Thr Ile Leu Gln Asn Asp His Ser Leu Leu Ala Ser Ser

100 105 110

Trp Ala Ser Ala Glu Leu His Ala Pro Pro Gly Ser Pro Gly Thr Ser

115 120 125

Ile Val Asn Leu Thr Cys Thr Thr Asn Thr Thr Glu Asp Asn Tyr Ser

130 135 140

Arg Leu Arg Ser Tyr Gln Val Ser Leu His Cys Thr Trp Leu Val Gly

145 150 155 160

Thr Asp Ala Pro Glu Asp Thr Gln Tyr Phe Leu Tyr Tyr Arg Tyr Gly

165 170 175

Ser Trp Thr Glu Glu Cys Gln Glu Tyr Ser Lys Asp Thr Leu Gly Arg

180 185 190

Asn Ile Ala Cys Trp Phe Pro Arg Thr Phe Ile Leu Ser Lys Gly Arg

195 200 205

Asp Trp Leu Ala Val Leu Val Asn Gly Ser Ser Lys His Ser Ala Ile

210 215 220

Arg Pro Phe Asp Gln Leu Phe Ala Leu His Ala Ile Asp Gln Ile Asn

225 230 235 240

Pro Pro Leu Asn Val Thr Ala Glu Ile Glu Gly Thr Arg Leu Ser Ile

245 250 255

Gln Trp Glu Lys Pro Val Ser Ala Phe Pro Ile His Cys Phe Asp Tyr

260 265 270

Glu Val Lys Ile His Asn Thr Arg Asn Gly Tyr Leu Gln Ile Glu Lys

275 280 285

Leu Met Thr Asn Ala Phe Ile Ser Ile Ile Asp Asp Leu Ser Lys Tyr

290 295 300

Asp Val Gln Val Arg Ala Ala Val Ser Ser Met Cys Arg Glu Ala Gly

305 310 315 320

Leu Trp Ser Glu Trp Ser Gln Pro Ile Tyr Val Gly Asn Asp Glu His

325 330 335

Lys Pro Leu Arg Glu Trp Phe Val Ile Val Ile Met Ala Thr Ile Cys

340 345 350

Phe Ile Leu Leu Ile Leu Ser Leu Ile Cys Lys Ile Cys His Leu Trp

355 360 365

Ile Lys Leu Phe Pro Pro Ile Pro Ala Pro Lys Ser Asn Ile Lys Asp

370 375 380

Leu Phe Val Thr Thr Asn Tyr Glu Lys Ala Gly Ser Ser Glu Thr Glu

385 390 395 400

Ile Glu Val Ile Cys Tyr Ile Glu Lys Pro Gly Val Glu Thr Leu Glu

405 410 415

Asp Ser Val Phe

420

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Trp Glu Arg Ser Leu Ala Gly Ala Glu Glu Thr Ile Pro Leu Gln Thr

1 5 10 15

Leu Arg Cys Tyr Asn Asp Tyr Thr Ser His Ile Thr Cys Arg Trp Ala

20 25 30

Asp Thr Gln Asp Ala Gln Arg Leu Val Asn Val Thr Leu Ile Arg Arg

35 40 45

Val Asn Glu Asp Leu Leu Glu Pro Val Ser Cys Asp Leu Ser Asp Asp

50 55 60

Met Pro Trp Ser Ala Cys Pro His Pro Arg Cys Val Pro Arg Arg Cys

65 70 75 80

Val Ile Pro Cys Gln Ser Phe Val Val Thr Asp Val Asp Tyr Phe Ser

85 90 95

Phe Gln Pro Asp Arg Pro Leu Gly Thr Arg Leu Thr Val Thr Leu Thr

100 105 110

Gln His Val Gln Pro Pro Glu Pro Arg Asp Leu Gln Ile Ser Thr Asp

115 120 125

Gln Asp His Phe Leu Leu Thr Trp Ser Val Ala Leu Gly Ser Pro Gln

130 135 140

Ser His Trp Leu Ser Pro Gly Asp Leu Glu Phe Glu Val Val Tyr Lys

145 150 155 160

Arg Leu Gln Asp Ser Trp Glu Asp Ala Ala Ile Leu Leu Ser Asn Thr

165 170 175

Ser Gln Ala Thr Leu Gly Pro Glu His Leu Met Pro Ser Ser Thr Tyr

180 185 190

Val Ala Arg Val Arg Thr Arg Leu Ala Pro Gly Ser Arg Leu Ser Gly

195 200 205

Arg Pro Ser Lys Trp Ser Pro Glu Val Cys Trp Asp Ser Gln Pro Gly

210 215 220

Asp Glu Ala Gln Pro Gln Asn Leu Glu Cys Phe Phe Asp Gly Ala Ala

225 230 235 240

Val Leu Ser Cys Ser Trp Glu Val Arg Lys Glu Val Ala Ser Ser Val

245 250 255

Ser Phe Gly Leu Phe Tyr Lys Pro Ser Pro Asp Ala Gly Glu Glu Glu

260 265 270

Cys Ser Pro Val Leu Arg Glu Gly Leu Gly Ser Leu His Thr Arg His

275 280 285

His Cys Gln Ile Pro Val Pro Asp Pro Ala Thr His Gly Gln Tyr Ile

290 295 300

Val Ser Val Gln Pro Arg Arg Ala Glu Lys His Ile Lys Ser Ser Val

305 310 315 320

Asn Ile Gln Met Ala Pro Pro Ser Leu Asn Val Thr Lys Asp Gly Asp

325 330 335

Ser Tyr Ser Leu Arg Trp Glu Thr Met Lys Met Arg Tyr Glu His Ile

340 345 350

Asp His Thr Phe Glu Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp Lys

355 360 365

Asp Ser Lys Thr Glu Thr Leu Gln Asn Ala His Ser Met Ala Leu Pro

370 375 380

Ala Leu Glu Pro Ser Thr Arg Tyr Trp Ala Arg Val Arg Val Arg Thr

385 390 395 400

Ser Arg Thr Gly Tyr Asn Gly Ile Trp Ser Glu Trp Ser Glu Ala Arg

405 410 415

Ser Trp Asp Thr Glu Ser His His His His His His

420 425

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Trp Tyr

20 25 30

Arg Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Ser Ser Gly Gly Phe Pro Tyr Tyr Asn Tyr Lys Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Tyr Asp Ser Phe Phe Asp Ile Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

<210> 8

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Gly Phe Thr Phe Pro Trp Tyr Arg Val His

1 5 10

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Ser Ile Arg Ser Ser Gly Gly Phe Pro Tyr Tyr Asn Tyr Lys Val Lys

1 5 10 15

Gly

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Phe Tyr Asp Ser Phe Phe Asp Ile

1 5

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Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

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Ala Ala Ser Ser Leu Gln Ser

1 5

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Gln Gln Ala Asn Ser Phe Pro Ile Thr

1 5

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Trp Tyr

20 25 30

Arg Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Arg Ser Ser Gly Gly Phe Pro Tyr Tyr Asn Tyr Lys Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Tyr Asp Ser Phe Phe Asp Ile Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 15

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic antibody sequences

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 327

<212> PRT

<213> artificial sequence

<220>

<223> synthetic antibody sequences

<400> 16

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 17

<211> 104

<212> PRT

<213> artificial sequence

<220>

<223> synthetic antibody sequences

<400> 17

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

1 5 10 15

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

20 25 30

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

35 40 45

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

50 55 60

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

65 70 75 80

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

85 90 95

Lys Ser Phe Asn Arg Gly Glu Cys

100

<210> 18

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of Vl

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105

Claims (27)

1. A method for preventing or treating acute respiratory syndrome (ARDS) in a subject, the method comprising administering to the subject a compound that binds to CD131 and neutralizes signaling through Interleukin (IL) 3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF).

2. The method of claim 1, wherein the ARDS is associated with one or more of:

a) Infection;

b) Sucking or sucking out foreign matter;

c) A physical trauma; and

d) Inflammatory diseases.

3. The method of claim 1 or claim 2, wherein the ARDS is associated with a viral infection.

4. A method according to any one of claims 1 to 3, wherein the ARDS is associated with a coronavirus infection.

5. The method of any one of claims 1-4, wherein the ARDS is associated with severe acute respiratory syndrome coronavirus (SARS-COV) infection.

6. The method of any one of claims 1-5, wherein the ARDS is associated with a SARS-CoV-2 infection.

7. The method of any one of claims 1 to 6, wherein the ARDS is associated with coronavirus disease 2019 (covd-19).

8. The method of any one of claims 1-7, wherein the subject has interstitial pneumonia.

9. The method of any one of claims 1 to 8, wherein administration of the compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF reduces or prevents an increase in one or more or all of:

a) The amount of neutrophils in the blood of the subject,

b) The amount of monocytes in the blood of the subject,

c) Neutrophil accumulation in the lungs of the subject,

d) Macrophage accumulation in the lungs of the subject,

e) Inflammation of the lungs of the subject,

f) Edema of the lungs of the subject,

g) NETosis in the lungs of the subject,

h) Myeloperoxidase activity in bronchoalveolar fluid of the lungs of the subject,

i) The amount of protein in the bronchoalveolar fluid of the lungs of the subject,

j) An amount of dsDNA in bronchoalveolar fluid of the lung of said subject, and

k) Lung injury score of the subject.

10. The method of any one of claims 1-9, wherein administering the compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF prevents a decrease in the percentage of blood oxygenation in the subject.

11. The method of any one of claims 1 to 10, wherein the compound that binds to CD131 and neutralizes signaling through IL-3, IL-5, and GM-CSF:

a) Inhibiting GM-CSF-induced proliferation of TF-1 cells with an IC50 of at least 100 nM; and/or

b) Inhibiting IL-5-induced proliferation of TF-1 cells with an IC50 of at least 100 nM; and/or

c) IL-3-induced proliferation of TF-1 cells was inhibited with an IC50 of at least 100 nM.

12. The method of any one of claims 1 to 11, wherein the compound that binds to CD131 and neutralizes signaling through IL-3, IL-5 and GM-CSF is a protein comprising an antigen binding site that binds to CD 131.

13. The method of claim 12, wherein when comprising SEQ ID NO:5 is immobilized on a solid surface and K _D K of the protein of the polypeptide, as determined by surface plasmon resonance _D About 10nM or less.

14. The method of any one of claims 1 to 13, wherein the compound that binds to CD131 and neutralizes signaling through IL-3, IL-5 and GM-CSF is a protein comprising Fv.

15. The method of claim 14, wherein the protein comprises:

(i) Single chain Fv fragments (scFv);

(ii) Dimeric scFv (di-scFv); or (b)

(iii) A double body;

(iv) A trisome;

(v) A tetrahedron;

(vi)Fab；

(vii)F(ab′) ₂ ；

(viii)Fv；

(ix) With the constant region, fc or heavy chain constant domain (C) _H ) 2 and/or C _H 3 to one of (i) to (viii);

(x) One of (i) to (viii) linked to albumin or a functional fragment or variant thereof or a protein that binds to albumin; or (b)

(xi) An antibody.

16. The method of claim 12 or claim 13, wherein the protein is a single domain antibody (sdAb).

17. The method of any one of claims 12 to 15, wherein the protein comprises an antibody variable region that binds to CD131 and competitively inhibits binding of antibody 9A2-VR24.29 to CD131, the antibody 9A2-VR24.29 comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:6 shown in the accompanying drawingsHeavy chain variable region (V) _H ) And a polypeptide comprising SEQ ID NO:18 (V _L )。

18. The method of any one of claims 12-167, wherein the antigen binding site binds to an epitope within site 2 of CD 131.

19. The method of any one of claims 12 to 18, wherein the antigen binding site binds to an epitope formed upon dimerization of two CD131 polypeptides.

20. The method of claim 19, wherein the antigen binding site binds to a residue within domain 1 of a CD131 polypeptide and a residue within domain 4 of another CD131 polypeptide.

21. The method of claim 20, wherein the residue within domain 1 of CD131 comprises the amino acid sequence of SEQ ID NO:1 and/or said residue within domain 4 of CD131 comprises the residue within the region 101-107 of SEQ ID NO: residues within the 364-367 region of 1.

22. A method according to any one of claims 12 to 15 or claims 17 to 21 wherein the protein comprises an antibody variable region comprising a VH comprising a sequence comprising SEQ ID NO:6, said VL comprising three CDRs comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:18, and the three CDRs of the VL of the amino acid sequence shown in seq id no.

23. The method of any one of claims 12 to 15 or claims 17 to 22, wherein the protein comprises a polypeptide comprising SEQ ID NO:6 and VH comprising the amino acid sequence set forth in SEQ ID NO:18, and a VL of the amino acid sequence shown in seq id no.

24. The method of any one of claims 12 to 15 or claims 17 to 23, wherein the protein comprises a polypeptide comprising SEQ ID NO:8 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain of the amino acid sequence shown in seq id no.

25. The method of any one of claims 1 to 24, wherein the compound that binds to CD131 is administered in combination with standard of care therapy.

26. The method of claim 25, wherein the standard of care regimen comprises one or more or all of the following:

a) A prone position;

b) Fluid management;

c) Administering nitric oxide;

d) Administering a neuromuscular blocking agent;

e) Artificial ventilation;

f) Oxygenation of the outer membrane; and

g) An antiviral agent or antibiotic is administered.

27. The method of claim 25, wherein the standard of care therapy comprises administration of adefovir (remdesired).