Methods for treating acute respiratory distress syndrome (ARDS) using mesenchymal precursors or stem cells

Abstract

The present disclosure relates to a method for treating or preventing acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject a composition comprising mesenchymal precursor or stem cells (MLPSCs).

Description

[Technical field]

[0001] The present disclosure relates to methods for treating or preventing Acute Respiratory Distress Syndrome (ARDS) in a subject in need thereof. [Background technology]

[0002] Respiratory diseases associated with various conditions, such as viral infections, are a problem in the general population. Often, these are accompanied by inflammation, which can worsen lung conditions and lead to acute respiratory distress syndrome (ARDS).

[0003] There remains an unmet therapeutic need in patients with acute respiratory distress syndrome (ARDS), particularly when secondary to viral infections, for which new treatment options are required. Summary of the Invention

[0004] The inventors have identified that corticosteroid treatment with dexamethasone in ARDS does not provide protection against death, particularly in patients under 65 years of age. The inventors have surprisingly identified that this deficiency is remedied by administering MLPSCs with dexamethasone. These findings suggest that treating ARDS patients with corticosteroids and MLPSCs may improve the treatment of ARDS, particularly in relation to survival prospects. Thus, in a first example, the disclosure relates to a method of treating or preventing acute respiratory distress syndrome (ARDS) in a human subject in need thereof, the method comprising administering to the subject a composition comprising a corticosteroid and mesenchymal precursor or stem cell (MLPSC). In one example, the subject is under 65 years of age.

[0005] The inventors have also identified an effective method of treating subjects with acute respiratory distress syndrome (ARDS) under the age of 65 by administering mesenchymal precursor or stem cells (MLPSCs) to these subjects. Thus, in another example, the present disclosure relates to a method of treating or preventing acute respiratory distress syndrome (ARDS) in a human subject in need thereof, the method comprising administering to the subject a composition comprising mesenchymal precursor or stem cells (MLPSCs), the subject being under the age of 65. In one example, the method further comprises administering a corticosteroid.

[0006] The inventors' findings suggest that subjects under 65 years of age may be selected for effective treatment according to the methods disclosed herein. Thus, in one example, the disclosure relates to a method of treating or preventing acute respiratory distress syndrome (ARDS) in a human subject in need thereof, the method comprising: selecting a subject with ARDS who is under 65 years of age; and administering to the subject a composition comprising mesenchymal precursor or stem cells (MLPSCs). In one example, a subject who is under 65 years of age and taking corticosteroids is selected.

[0007] In the above example, the subject may be under 60 years old. In another example, the subject may be between 18 and 65 years old. In another example, the subject may be between 18 and 60 years old.

[0008] In one example, the subject's ARDS is moderate or severe.

[0009] In one example, the subject is taking a corticosteroid prior to administration of the cell compositions disclosed herein. In one example, the corticosteroid is dexamethasone.

[0010] In one example, the subject is on a ventilator. For example, the subject can be mechanically ventilated prior to administering the MLPSCs. In one example, the subject is weaned off the ventilator following treatment. In one example, the subject is weaned off the ventilator within 60 days of treatment.

[0011] In one example, ARDS is caused by a viral infection, which may be caused by, for example, a rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus.

[0012] In one example, ARDS is caused by a coronavirus infection. The coronavirus can be, for example, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), COVID-19, 229E, NL63, OC43, or KHU1. In one example, the coronavirus is SARS-CoV, MERS-CoV, or COVID-19 (SARS-CoV-2).

[0013] In one example, ARDS is caused by thrombosis, such as venous thrombosis or arterial thrombosis, hi another example, ARDS is caused by pulmonary embolism.

[0014] In one example, the treated subject's risk of death is reduced following treatment. In one example, the treated subject's risk of death is reduced by 30-60%. In one example, the treated subject has improved 60-day survival.

[0015] In one example, improved survival is an increase in days of survival on ventilation. Thus, in one example, a reduced risk of death is determined in subjects breathing unassisted by mechanical ventilation.

[0016] In one example, treatment according to the present disclosure increases ventilation-free survival days, in one example, the increase is observed 60 days after treatment.

[0017] In one example, the treatment improves respiratory function. In one example, improved respiratory function is defined as resolution and / or improvement of ARDS as defined by the Berlin Criteria on one or more or all of the following days: 7, 14, 21, and 30 after treatment. In one example, the improvement is observed on day 7. In one example, the improvement is observed on day 14. For example, the treatment may improve respiratory function as defined by the Berlin Criteria on day 14 and / or day 21. In one example, the improved respiratory function is maintained beyond day 7 as compared to baseline respiratory function. In one example, the improved respiratory function is maintained on day 14 as compared to baseline respiratory function.

[0018] In one example, the treatment improves clinical outcomes. In one example, the improvement in clinical outcomes is assessed based on a 7-point ordinal scale at baseline and one or more or all of days 7, 14, 21, and 30, and at discharge.

[0019] In another example, the treatment reduces the level of at least one inflammatory biomarker(s) compared to baseline, and at least one inflammatory biomarker(s) is - Decreased influx of neutrophils and macrophages into the lungs, - Reduction of inflammasomes, -Reduction in macrophage activation and neutrophil migration to the lungs, - Decreased T cell influx and activation, or -Shows a reduction in circulating biomarkers of macrophage and neutrophil inflammation.

[0020] In one example, the inflammatory biomarker(s) include: - CXCR3 binding chemokines, preferably CXCL10 and / or CXCL9, - CCR2-binding chemokines, preferably CCL2, CCL3, and / or CCL7; -IL-6, -IL-8, -TNF, -IL-18, -CCL19, -IL-4, -IL-13, -GM-CSF, -CRP, or Ferritin is one or more of the following:

[0021] In one example, the treatment reduces CRP and / or ferritin levels within 3 to 14 days of administering the MLPSCs.

[0022] In one example, the MLPSCs have been cryopreserved and thawed.

[0023] In one example, the MLPSCs are culture-expanded from an intermediate cryopreserved MLPSC population. In another example, the MLPSCs are culture-expanded for at least about 5 passages. In one example, the MLPSCs express at least 13 pg of TNF-R1 per million MLPSCs. In one example, the MLPSCs express about 13 pg to about 44 pg of TNF-R1 per million MLPSCs. In one example, the culture-expanded MLPSCs are culture-expanded for at least 20 population doublings. In another example, the culture-expanded MLPSCs are culture-expanded for at least 30 population doublings. In one example, the MLPSCs are mesenchymal stem cells (MSCs). In another example, the MLPSCs are allogeneic. For example, the MLPSCs can be allogeneic MSCs.

[0024] In another example, the MLPSCs are engineered to carry or express an antiviral or thrombolytic agent. In one example, the antiviral agent is Remdesivir. In one example, the thrombolytic agent is selected from the group consisting of Eminase (anistreplase), Retavase, and Streptase (streptokinase, kabikinase).

[0025] In another example, the MLPSCs are genetically modified to express an antiviral peptide or a nucleic acid encoding same.

[0026] In one example, the composition is administered intravenously.

[0027] In one example, the method of the present disclosure 7 ~2×10 8 For example, administering 1×10 cells. 7 ~2×10 8 Multiple doses of cells may be administered on days 0, 30, 60, and 90. In one example, the methods of the disclosure provide for administration of about 1×10 cells per dose. 8 In one example, the subject is administered two doses of the cells.

[0028] In one example, the subject receives the second dose within 7 days of the first dose being administered. In one example, the second dose is administered 4 days after the first dose. In one example, the dose is 2×10 per kilogram of body weight. 6 Contains cells.

[0029] In another example, the composition further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).In one example, the composition further comprises a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), wherein the HSA solution comprises 5% HSA and 15% buffer.

[0030] In one example, the composition is 6.68 x 10 6 Contains > 10 viable cells / mL. [Brief description of the drawings]

[0031] [Figure 1] Cell therapy offers some degree of protection against death at 60 days in all patients. A: All intention to treat (ITT) patients, B: All per protocol (PP) patients. [Diagram 2] Cell therapy provides protection against 60-day mortality in patients <65 years of age. A: ITT patients <65 years of age, B: ITT patients ≥65 years of age. [Diagram 3]Cell therapy provides protection against 60-day mortality in patients <65 years of age. A: PP patients <65 years of age (n=123), B: PP patients ≥65 years of age (n=94). [Figure 4] Cell therapy protects against all-cause mortality at day 60 in ITT and PP patients aged <60 years who received dexamethasone at baseline. A: ITT patients, B: PP patients. [Diagram 5] Baseline dexamethasone does not provide protection against death at day 60 in control patients. A: ITT patients aged <65 years, B: ITT patients aged ≥65 years. [Figure 6A] Baseline dexamethasone provides synergistic protection against 60-day mortality when combined with cell therapy in patients <65 years of age. Furthermore, cell therapy plus dexamethasone outperforms all other treatment arms in reducing mortality over 60 days. ITT patients. [Figure 6B] Baseline dexamethasone provides synergistic protection against 60-day mortality when combined with cell therapy in patients <65 years of age. Furthermore, cell therapy plus dexamethasone outperforms all other treatment arms in reducing mortality over 60 days. PP patients. [Figure 6C] Baseline dexamethasone provides synergistic protection against 60-day mortality when combined with cell therapy in patients <65 years of age. Furthermore, cell therapy plus dexamethasone outperforms all other treatment arms in reducing mortality over 60 days. All treated patients <65 years of age on dexamethasone (n=73). [Figure 7]Cell therapy plus dexamethasone: Analysis of respiratory function and clinical improvement in a discovery population <65 years old. Respiratory function improvement was measured as resolution and / or improvement of ARDS defined by the Berlin criteria at days 7, 14, 21, and 30 after randomization, and clinical improvement was assessed based on a 7-point ordinal scale at baseline and days 7, 14, 21, and 30 and at discharge. A: Improvement in respiratory function in ITT patients <65 years old (n=73) on dexamethasone. B: Clinical improvement in ITT patients <65 years old (n=73) on dexamethasone. [Figure 8] Analysis of respiratory function improvement in A: patients <65 years old and B: patients ≥65 years old. Respiratory function measured as resolution and / or improvement of ARDS defined by the Berlin criteria on days 7, 14, 21, and 30 after randomization. [Figure 9] Cell therapy reduces mortality by 48% over 90 days in a prespecified analysis of patients <65 years (n=123). [Figure 10] Cell therapy increases 60-day ventilator-free survival in patients <65 years of age. A) All treated patients <65 years of age (n=123). B) All treated patients <65 years of age taking dexamethasone (n=73). [Figure 11A] CRP levels at baseline and on days 3, 7, and 14. [Figure 11B] Ferritin levels at baseline and on days 3, 7, and 14. [Figure 11C] D-dimer at baseline and days 3, 7, and 14. [Figure 12] Patients >65 years old have higher levels of baseline inflammation. Data are fold change in levels. [Figure 13] Stratified analysis of inflammatory biomarkers by age group. Data are fold change in levels from baseline. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions shall be taken to encompass one and several (i.e., one or more) of that step, composition, group of steps or group of compositions.

[0033] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and improvements other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations, or any two or more of such steps or features.

[0034] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure, as described herein.

[0035] Any example disclosed herein is intended to apply mutatis mutandis to any other example, unless specifically stated otherwise.

[0036] Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, stem cell therapy, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0037] Unless otherwise indicated, the surgical techniques utilized in this disclosure are standard procedures well known to those skilled in the art.

[0038] Methods for obtaining and enriching populations of mesenchymal stem or progenitor cells are known in the art. For example, enriched populations of mesenchymal stem or progenitor cells can be obtained by the use of flow cytometry and cell sorting procedures based on the use of cell surface markers expressed on mesenchymal stem or progenitor cells.

[0039] All documents cited or referenced herein, and all documents cited or referenced within the documents cited herein, are hereby incorporated by reference in their entirety, along with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein.

[0040] Selected Definitions The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is deemed to provide clear support for both meanings or either meaning.

[0041] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise stated.

[0042] The terms "level" and "amount" are used to define the amount of a particular substance in a cell preparation. For example, a particular concentration, weight, percentage (e.g., v / v%) or ratio can be used to define the level of a particular substance. In one example, the level is expressed in terms of how much of a particular marker is expressed by the cells of the present disclosure under culture conditions. In one example, the expression refers to cell surface expression. In another example, the level is expressed in terms of how much of a particular marker is released from the cells described herein under culture conditions.

[0043] In one example, the levels are expressed in pg / ml. 6 Expressed in pg per cell. Levels of pg / ml can be adjusted to 10 or 20 as needed. 6For example, in the context of TNF-R1, 200 pg / ml of TNF-R1 is equivalent to 10 6 In one example, 225 pg / ml of TNF-R1 corresponds to approximately 23.5 pg of TNF-R1 per cell. 6 This corresponds to approximately 26.5 pg of TNF-R1 per cell. 6 In another example, 260 pg / ml of TNF-R1 corresponds to approximately 27 pg of TNF-R1 per 10 cells. 6 In another example, 270 pg / ml of TNF-R1 corresponds to approximately 30 pg of TNF-R1 per cell. 6 This corresponds to approximately 32 pg of TNF-R1 per cell.

[0044] In one example, the level of a particular marker is determined under culture conditions. The term "culture conditions" is used to refer to cells growing in culture. In one example, culture conditions refer to an actively dividing population of cells. Such cells may be in an exponential growth phase, in one example. For example, the level of a particular marker may be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker may be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. One of skill in the art will appreciate that secreted markers may be measured by sampling the culture medium, while cell surface expressed markers may be measured by evaluating a sample of the cell lysate. In one example, the sample is taken when the cells are in an exponential growth phase. In one example, the sample is taken after at least two days in culture.

[0045] Culturing to expand cells from a cryopreserved intermediate refers to thawing cells that have been subjected to deep freezing and in vitro culture under conditions suitable for cell growth.

[0046] In one example, the "level" or "amount" of a particular marker, such as TNF-R1, is determined after the cells are cryopreserved and then reseeded in culture. For example, the level is determined after a first cryopreservation of the cells. In another example, the level is determined after a second cryopreservation of the cells. For example, the cells may be expanded in culture from a cryopreserved intermediate and cryopreserved a second time before being reseeded in culture so that the level of the particular marker can be determined under culture conditions.

[0047] Throughout this specification the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean 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.

[0048] As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context dictates otherwise.

[0049] "Isolated" or "purified" refers to a cell that has been separated from at least some components of its natural environment. The term includes total physical separation of the cell from its natural environment (e.g., removal from a donor). The term "isolated" includes a change in the relationship of the cell to adjacent cells with which it is directly associated, for example, by dissociation. The term "isolated" does not refer to cells in a tissue section. When used to refer to a population of cells, the term "isolated" includes a population of cells resulting from propagation of an isolated cell of the present disclosure.

[0050] The terms "passage," "passaging," or "subculture" are used in the context of this disclosure to refer to known cell culture techniques used to keep cells alive and growing under culture conditions for extended periods of time so that cell numbers can be continually increased. The degree of subculture that a cell line has undergone is often expressed as a "passage number," which is generally used to refer to the number of times the cells have been subcultured. In one example, one pass involves removing non-adherent cells and leaving adherent mesenchymal precursors or stem cells. Such mesenchymal precursors or stem cells can then be dissociated from the substrate or flask (e.g., by using a protease such as trypsin or collagenase), medium can be added, optional washing (e.g., by centrifugation) can be performed, and the mesenchymal precursors or stem cells can then be re-plated or reseeded into one or more culture vessels that contain a larger overall surface area. The mesenchymal precursors or stem cells can then continue to grow in culture. In another example, a method for removing non-adherent cells includes a non-enzymatic treatment step (e.g., with EDTA). In one example, the mesenchymal precursors or stem cells are passaged at or near confluence (e.g., about 75% to about 95% confluence). In one example, the mesenchymal precursors or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells / ml of culture medium.

[0051] The term "medium" (singular or plural), as used in the context of this disclosure, includes components of the environment surrounding cells in culture. It is envisioned that the medium contributes to and / or provides conditions suitable for allowing cell growth. The medium may be a solid, liquid, gas, or a mixture of phases and materials. The medium may include liquid growth media as well as liquid media that do not support cell growth. Exemplary gaseous media include the gaseous phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0052] As used herein, the terms "treating", "treat" or "treatment" include administering a population of mesenchymal stem cells or precursor cells and / or their progeny and / or soluble factors derived therefrom, thereby reducing or eliminating at least one symptom of ARDS. In one example, the treatment includes administering a population of culture-expanded mesenchymal stem cells or precursor cells. In one example, the treatment response is determined relative to a baseline. In one example, the treatment response is determined relative to a target patient population. In one example, the treatment improves the subject's ARDS from severe to moderate.

[0053] In one example, the method of the present disclosure inhibits disease progression or disease complications in a subject. "Inhibiting" disease progression or disease complications in a subject means preventing or reducing disease progression and / or disease complications in a subject. Thus, in one example, the method of the present disclosure inhibits the progression of ARDS severity.

[0054] As used herein, the term "prevent" or "preventing" includes administering a population of mesenchymal stem or precursor cells and / or their progeny and / or soluble factors derived therefrom, thereby arresting or inhibiting the development of at least one symptom of ARDS.

[0055] As used herein, the term "subject" refers to a human subject. For example, a subject may be an adult. Terms such as "subject," "patient," or "individual" are terms that may be used interchangeably in this disclosure, where context permits.

[0056] The term "thrombosis" is used herein to refer to the formation of a blood clot or clot. In one example, thrombosis is "arterial thrombosis," where a clot develops in an artery. Such clots are particularly dangerous to a subject because they can block blood flow to major organs such as the heart or brain. In one example, thrombosis is "venous thrombosis," where a clot develops in a vein.

[0057] The term "pulmonary embolism" is used herein to refer to the blockage of an artery in the lungs by material that has traveled through the bloodstream from elsewhere in the body.

[0058] As used herein, the term "genetically unmodified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursor or stem cells transfected with a nucleic acid encoding Ang1 are considered to be genetically modified.

[0059] "C-reactive protein" or "CRP" is an inflammatory mediator whose levels are elevated under conditions of acute inflammatory flare-ups and rapidly normalize once inflammation subsides. Circulating CRP levels can be measured in plasma samples to provide a measure of inflammation in a subject.

[0060] The term "total dose" is used in the context of this disclosure to refer to the total number of cells received by a subject treated according to the present disclosure. In one example, the total dose consists of one administration of cells. In another example, the total dose consists of two administrations of cells. In another example, the total dose consists of three administrations of cells. In another example, the total dose consists of four or more administrations of cells. For example, the total dose can consist of two to four administrations of cells.

[0061] The term "clinically proven" (used independently or to modify the term "effective") means that the effectiveness has been proven by a clinical trial that meets the approval standards of the US Food and Drug Administration, EMEA, or the corresponding national regulatory agency. For example, the clinical study may be a randomized, double-blind study of appropriate size that is used to clinically prove the effectiveness of the composition. In one example, the clinically proven effective amount is the amount shown by a clinical trial to meet a particular endpoint. In one example, the endpoint is protection against death.

[0062] Thus, the terms "clinically proven effective" and "clinically proven effective" may be used in the context of the present disclosure to refer to a dose, dosing regimen, treatment, or method disclosed herein. Effectiveness can be measured based on changes in the course of the disease in response to administering a composition disclosed herein. For example, a composition of the present disclosure is administered to a subject in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of ARDS. To determine whether the amount and time of treatment is sufficient, various indicators reflecting the severity of ARDS can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity or symptoms. In one example, the degree of improvement is determined by a physician who can make this determination based on signs, symptoms, or other test results. In one example, a clinically proven effective amount improves patient survival. In another example, a clinically proven effective amount reduces the subject's risk of death. In another example, a clinically proven effective amount reduces the subject's circulating CRP level.

[0063] Acute respiratory distress syndrome (ARDS) The disclosed method relates to the treatment of acute respiratory distress syndrome (ARDS) by administering a composition disclosed herein. In one example, the method includes administering a composition comprising MLPSCs. Thus, in one example, the composition can include MSCs. In one example, the disclosed method includes administering a cell composition disclosed herein, such as a composition comprising MLPSCs, and a corticosteroid. In this example, the corticosteroid can be administered simultaneously or sequentially with the cell composition. In one example, the subject is pre-administered a corticosteroid prior to administering the cell composition disclosed herein. In this example, the corticosteroid can continue to be administered with the cell composition.

[0064] In one example, the corticosteroid is a long-acting or intermediate-acting (half-life < 36 hours) corticosteroid. In one example, the corticosteroid is a long-acting (half-life of 36 - 72 hours). In one example, the corticosteroid is dexamethasone. Other examples of corticosteroids include prednisone and methylprednisolone.

[0065] The term "acute respiratory distress syndrome (ARDS)" is a type of respiratory failure characterized by widespread inflammation of the lungs, poor oxygenation, and a non-compliant or "stiff" lung. This disorder is generally associated with capillary endothelial injury and diffuse alveolar damage.

[0066] In one example, the method of the present disclosure prevents or treats a subject having mild ARDS. In another example, the method of the present disclosure prevents or treats a subject having moderate ARDS. In another example, the method of the present disclosure prevents or treats a subject having severe ARDS. In another example, the method of the present disclosure prevents or treats a subject having moderate or severe ARDS. In one example, the method of the present disclosure treats a subject having ARDS who requires ventilation. For example, the subject can be attached to a mechanical ventilator.

[0067] In one example, the severity of ARDS is diagnosed according to the PaO2 / FiO2 ratio. For example, the severity of ARDS can be diagnosed as follows: (mild: 26.6 kPa < PaO2 / FiO2 ≤ 39.9 kPa, moderate: 13.3 kPa < PaO2 / FiO2 ≤ 26.6 kPa, severe: PaO2 / FiO2 ≤ 13.3 kPa). In one example, the severity of ARDS can be diagnosed according to the Berlin definition, as summarized in the following table.

Table 1

[0068] In another example, the severity of ARDS can be diagnosed as follows: mild (PaO2 / FiO2 200-300 mmHg), moderate (PaO2 / FiO2 100-200 mmHg), and severe (PaO2 / FiO2 less than 100 mmHg).

[0069] In one example, a subject with moderate to severe ARDS has a circulating CRP level >4 mg / L.

[0070] In one example, the subject treated according to the methods of the present disclosure is under 65 years of age. In another example, the subject is under 60 years of age. In one example, the subject is at least 18 years of age. In one example, the subject is between 18 and 65 years of age. In another example, the subject is between 18 and 60 years of age.

[0071] In another example, the subject treated according to the disclosed methods is taking a corticosteroid. For example, the subject may be taking dexamethasone. In one example, the subject is under 65 years old and is taking a corticosteroid, such as dexamethasone. In one example, the subject is under 60 years old and is taking a corticosteroid, such as dexamethasone. In one example, the corticosteroid is a long-acting or intermediate-acting (half-life <36 hours) corticosteroid. In one example, the corticosteroid is a long-acting (half-life 36-72 hours). In one example, the corticosteroid is dexamethasone. Other examples of corticosteroids include prednisone and methylprednisolone.

[0072] In another example, the disclosed method includes administering a cellular composition disclosed herein and a corticosteroid. In this example, the corticosteroid can be administered simultaneously or sequentially with the cellular composition. In one example, the subject is pre-administered a corticosteroid prior to administering the cellular composition disclosed herein. In this example, the corticosteroid can continue to be administered along with the cellular composition.

[0073] In one example, the disclosed method includes selecting a subject under 65 years of age for treatment. In another example, the disclosed method includes selecting a subject under 65 years of age who is taking a corticosteroid for treatment. In another example, the disclosed method includes selecting a subject under 65 years of age who is taking dexamethasone for treatment. In one example, the subject is selected based on an appropriate questionnaire or series of questions from the treating clinician, for example, inquiring about the subject's age and current medications.

[0074] Subjects treated according to the present disclosure may have symptoms indicative of ARDS. Exemplary symptoms may include fatigue, dyspnea, shortness of breath, inability to exercise or decreased ability to exercise, coughing with or without blood or mucus, pain when breathing in or out, wheezing, chest tightness, unexplained weight loss, musculoskeletal pain, rapid breathing (tachypnea), and pale skin (cyanosis).

[0075] In another example, the subject has pneumonia.

[0076] In another example, the subject has ARDS secondary to a viral infection. In one example, the subject's ARDS is secondary to an infection with rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, the subject's ARDS is secondary to an infection with coronavirus. For example, the subject's ARDS can be secondary to an infection with SARS-CoV, MERS-CoV, or COVID-19.

[0077] In one example, the subject has one or more of myocarditis, pericarditis, or valvulitis. In one example, the subject has virally-induced myocarditis, pericarditis, or valvulitis. For example, the subject may have viral myocarditis.

[0078] In one example, ARDS is caused by a viral infection. For example, ARDS can be caused by a rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, ARDS can be caused by a coronavirus. For example, the coronavirus can be SARS-CoV, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), or COVID-19. In one example, ARDS is caused by an Epstein-Barr virus (EBV) or herpes simplex virus (HSV).

[0079] In another example, ARDS is caused by thrombosis. In another example, ARDS is caused by embolism. In one example, ARDS is caused by pulmonary embolism.

[0080] In another example, ARDS is secondary to hemophagocytic lymphohistiocytosis (HLH). HLH is a life-threatening disease characterized by lymphocyte and macrophage hyperinflammation. HLH can be caused by viral infections such as EBV, CMV, HHV, etc. Thus, in one example, HLH is secondary or acquired HLH. For example, HLH can be secondary to viral infections and lead to the development of ARDS in subjects.

[0081] In one example, the treatment protects against death or confers improved survival. In one example, the protection against death is determined 60 days after treatment. In another example, the protection against death is determined 50-70 days after treatment. For example, the treated subject's risk of death can be reduced after treatment. In one example, the treated subject's risk of death is reduced by 30-60%. In one example, the treated subject's risk of death is reduced by 40-50%. In one example, the treated subject's risk of death is reduced by at least 30%. In one example, the treated subject's risk of death is reduced by at least 40%.

[0082] In one example, treatment with the method of the present disclosure reduces the risk of thrombosis in a subject.In one example, the risk of the subject is reduced compared to a subject not receiving treatment.In one example, treatment reduces the risk of thrombosis, which is arterial thrombosis.Thus, in one example, treatment reduces the risk of heart attack or stroke in patients with ARDS.

[0083] In one example, the treatment reduces the subject's CRP level. In one example, the treatment reduces CRP by at least 100 mg / dl compared to baseline. In another example, the treatment reduces CRP by at least 150 mg / dl compared to baseline.

[0084] In one example, the treatment reduces the subject's circulating CRP level to 80 mg / dl or less. In one example, the treatment reduces the subject's circulating CRP level to 60 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 50 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 30 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 40 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 20 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 10 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 5 mg / dl or less. In another example, the treatment reduces the subject's circulating CRP level to 3 mg / dl or less.

[0085] In another example, the treatment reduces CRP to between 0.5 mg / dl and 30 mg / dl. In another example, the treatment reduces CRP to between 0.5 mg / dl and 10 mg / dl. In one example, the treatment reduces CRP and / or ferritin levels within 3-14 days of administering the MLPSCs.

[0086] In another example, the disclosure encompasses selecting a subject having ARDS for treatment. In one example, the subject has moderate or severe ARDS. In one example, the method includes selecting a subject having ARDS who is under 65 years old. In one example, the method includes selecting a subject who is under 65 years old and taking corticosteroids. In one example, the selected subject is treated according to the methods disclosed herein.

[0087] Inflammatory Biomarkers In one example, the treatment reduces the level(s) of inflammatory biomarker(s) in the subject. In one example, the reduction in inflammatory biomarkers indicates a decrease in neutrophil and macrophage influx into the lung, e.g., CCR2-binding chemokines such as CCL2, CCL3, and CCL7. In one example, the subject has a reduction in levels of CCL2. In another example, the subject has a reduction in levels of CCL3. In another example, the subject has a reduction in levels of CCL7. In another example, the inflammatory biomarkers are CXCR3-binding chemokines such as CXCL10 and CXCL9. In one example, the subject has a reduction in levels of CXCL10. In another example, the subject has a reduction in levels of CXCL9.

[0088] In another example, the reduction in inflammatory biomarkers indicates a reduction in inflammasomes. Inflammasomes are stimulus-induced cytoplasmic multimeric protein complexes. In another example, the reduction in inflammatory biomarkers indicates a reduction in macrophage activation and neutrophil homing to the lung. Examples of inflammatory biomarkers that, when reduced, indicate a reduction in inflammasomes and a reduction in macrophage activation / neutrophil homing to the lung are IL-6, IL-8, tumor necrosis factor (TNF), and interleukin-18 (IL-18). In one example, the subject has a reduction in the level of IL-6. In another example, the subject has a reduction in the level of IL-8. In another example, the subject has a reduction in the level of TNF. In another example, the subject has a reduction in the level of IL-18.

[0089] In another example, a decrease in inflammatory biomarkers indicates decreased T cell influx and activation. Examples of inflammatory biomarkers that, when decreased, indicate decreased T cell influx and activation are CC motif chemokine ligand 19 (CCL19), interleukin-4 (IL-4), interleukin-13 (IL-13), and granulocyte-macrophage colony stimulating factor (GM-CSF). In one example, the subject has a decreased level of CCL19. In another example, the subject has a decreased level of IL-4. In another example, the subject has a decreased level of IL-13. In another example, the subject has a decreased level of GM-CSF.

[0090] In another example, the reduction in inflammatory biomarkers indicates a decrease in circulating biomarkers of macrophage and neutrophil inflammation, such as CRP, ferritin, or D-dimer.In one example, the reduced circulating biomarker is "C-reactive protein" or "CRP".CRP is an inflammatory mediator whose levels are elevated under conditions of acute inflammatory recurrence and rapidly normalize when inflammation subsides.Circulating CRP levels can be measured in plasma samples to provide a measure of inflammation in a subject.

[0091] In one example, the treatment reduces the subject's CRP level. In one example, the treatment reduces CRP by at least 100 mg / dl compared to baseline. In another example, the treatment reduces CRP by at least 150 mg / dl compared to baseline. In one example, the treatment reduces the subject's CRP level by about 0.1-fold. In another example, the treatment reduces the subject's CRP level by about 0.2-fold. In another example, the treatment reduces the subject's CRP level by about 0.3-fold. In another example, the treatment reduces the subject's CRP level by about 0.4-fold. In another example, the treatment reduces the subject's CRP level by about 0.5-fold. In another example, the treatment reduces the subject's CRP level by 0.6-fold. In these examples, the treatment reduces the subject's CRP level by a fold change compared to the subject's baseline CRP level.

[0092] In another example, the decreased circulating biomarker is ferritin. Ferritin is a blood protein that contains iron. Ferritin levels can be measured in blood samples to provide a measure of the subject's iron level. In one example, the treatment reduces the subject's ferritin level. In one example, the treatment reduces the subject's ferritin level by about 0.1-fold. In another example, the treatment reduces the subject's ferritin level by about 0.2-fold. In another example, the treatment reduces the subject's ferritin level by about 0.3-fold. In another example, the treatment reduces the subject's ferritin level by about 0.4-fold. In another example, the treatment reduces the subject's ferritin level by about 0.5-fold. In these examples, the treatment reduces the subject's ferritin level by a fold change compared to the subject's baseline ferritin level.

[0093] Methods for determining the level of the inflammatory biomarkers disclosed herein are known in the art. In one example, the level of a particular marker is determined in a sample (e.g., a blood sample, a plasma sample, or a serum sample) obtained from a patient or subject. For example, the level of the inflammatory biomarker according to the present disclosure is determined in a plasma sample. In another example, the level of the inflammatory biomarker is determined in a serum sample.

[0094] In one example, the level of the inflammatory biomarker is determined by measuring the level of protein expression in a sample obtained from the subject. For example, the inflammatory biomarker level can be measured in the sample using an antibody-based immunoassay, such as an enzyme-linked immunosorbent (ELISA) assay, a multiplex immunoassay, such as a Luminex assay (see, e.g., Cook et al. Methods. 158:27-32.2019), or a fluorescent bead-based immunoassay. In these examples, the level of the inflammatory biomarker is expressed in pg / mL. In another example, the level of the inflammatory biomarker is expressed as a fold change compared to an appropriate control. In another example, the level of the inflammatory biomarker is determined by measuring the level of gene expression in a sample obtained from the subject. For example, the inflammatory biomarker level can be measured in the sample using a molecular-based assay, such as a qualitative polymerase chain reaction (PCR)-based assay, or a multiplex PCR assay, such as a Luminex assay (see, e.g., Cook et al. Methods. 158:27-32.2019). In one example, the level of gene expression of the inflammatory biomarker is expressed as a fold change compared to an appropriate control, e.g., the fold change is calculated as log2(fold-change).

[0095] In one example, the levels of multiple inflammatory biomarkers are measured in a single sample. In another example, the levels of multiple inflammatory biomarkers are measured in separate samples. In one example, the levels of the biomarkers are measured before treatment with MLPSCs. In another example, the levels of the biomarkers are measured after treatment with MLPSCs. In another example, the levels of the biomarkers are measured at baseline and monitored over time to determine if the subject requires more intensive or longer-term dosing.

[0096] The level of inflammatory biomarkers can be compared between samples to determine whether the level of inflammatory biomarkers is reduced.In these examples, the samples can be evaluated to determine whether inflammation is reduced and whether the reduction in inflammation is sustained.In one example, the sustained reduction in inflammation is determined based on the observed reduction in inflammation from baseline in at least two samples after administration of cell therapy.

[0097] Mesenchymal precursor cells The term "mesenchymal precursor or stem cell (MLPSC)" as used herein refers to undifferentiated multipotent cells that have the ability to self-renew while maintaining pluripotency and differentiate into several cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or non-mesodermal origin, such as hepatocytes, neural cells, and epithelial cells. For the avoidance of doubt, "mesenchymal precursor cells" refers to cells that can differentiate into mesenchymal cells such as bone, cartilage, muscle and adipocytes, and fibrous connective tissue.

[0098] The term "mesenchymal precursor or stem cell" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0099] Mesenchymal precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or homogeneous. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or homogeneous cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0100] In one example, the mesenchymal precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal precursors or stem cells are expanded in culture and cryopreserved.

[0101] Mesenchymal precursors or stem cells are primarily present in bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ lineages such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal precursors or stem cells can differentiate into a number of cell types, including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage commitment and differentiation pathways that these cells enter depend on various influences from mechanical influences and / or endogenous bioactive factors such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0102] The terms "enriched", "enriched", or variations thereof, are used herein to describe a cell population in which the percentage of one particular cell type or the percentage of multiple particular cell types is increased when compared to an untreated population of cells (e.g., cells in their natural environment). In one example, a population enriched for mesenchymal precursors or stem cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% mesenchymal precursors or stem cells. In this regard, the term "a population of cells enriched for mesenchymal precursors or stem cells" is used to give explicit support to the term "a population of cells comprising X% mesenchymal precursors or stem cells", where X% is a percentage described herein. Mesenchymal precursors or stem cells, in some instances, can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0103] In one example of the present disclosure, the mesenchymal precursors or stem cells are mesenchymal stem cells (MSCs). The MSCs may be a homogenous composition or a mixed cell population enriched in MSCs. The homogenous MSC composition may be obtained by culturing adherent bone marrow or periosteal cells, and the MSCs may be identified by specific cell surface markers identified with unique monoclonal antibodies. Methods for obtaining cell populations enriched in MSCs are described, for example, in U.S. Pat. No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0104] In another example, the mesenchymal precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0105] In one example, mesenchymal precursors or stem cells are culture-expanded from a population of MSCs that express markers including CD73, CD90, CD105, and CD166, and lack expression of hematopoietic cell surface antigens such as CD45 and CD31. For example, mesenchymal precursors or stem cells can be culture-expanded from a population of MSCs that are CD73+, CD90+, CD105+, CD166+, CD45-, and CD31-. In one example, the population of MSCs is further characterized by low levels of major histocompatibility complex (MHC) class I. In another example, the MSCs are negative for major histocompatibility complex class II molecules and negative for costimulatory molecules CD40, CD80, and CD86. In one example, the culture-expansion includes 5 passages.

[0106] In one example, mesenchymal precursor or stem cells are CD105+, CD156+, and CD45-. In another example, mesenchymal or precursor cells also express TNFR1 and suppress IL-2Rα expression on activated lymphocytes.

[0107] The isolated or enriched mesenchymal precursors or stem cells can be expanded in vitro by culture. The isolated or enriched mesenchymal precursors or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0108] In one example, isolated or enriched mesenchymal precursors or stem cells are cultured in culture medium (serum-free or serum-supplemented), such as alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at 50,000 viable cells / cm. 2 Cells are seeded at 37 °C and allowed to attach to the culture vessel overnight at 37 °C and 20% O. The culture medium is then exchanged and / or changed as necessary, and the cells are cultured for an additional 68-72 h at 37 °C and 5% O.

[0109] As will be appreciated by those skilled in the art, cultured mesenchymal precursors or stem cells are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal precursors or stem cells are also biologically distinct from in vivo cells, having a higher proliferation rate compared to most non-cycling (quiescent) cells in vivo.

[0110] In one example, a population of cells is enriched from a cell preparation that contains STRO-1+ cells in a selectable morphology. In this context, the term "selectable morphology" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., mesenchymal precursor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1 bright). Thus, indicating that a cell is STRO-1+ does not mean that the cell is selected solely by STRO-1 expression. In one example, the cell is selected based on at least STRO-3 expression, e.g., STRO-3+ (TNAP+).

[0111] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected, isolated, or enriched from a wide variety of sources. That said, in some instances, these terms indicate support for selection from any tissue that comprises STRO-1+ cells (e.g., mesenchymal precursor cells) or vascular tissue or pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues described herein.

[0112] In one example, the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0113] "Individually" means that the present disclosure encompasses each listed marker or group of markers individually, and notwithstanding that each marker or group of markers may not be individually listed herein, the appended claims may define such markers or groups of markers individually and divisibly from one another.

[0114] "Collectively" means that the disclosure encompasses any number or combination of the recited markers or groups of markers, and notwithstanding that such numbers or combinations of markers or groups of markers may not be specifically recited herein, the appended claims may define such combinations or subcombinations separately and divisibly from any other combinations of the markers or groups of markers.

[0115] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, as used herein, TNAP refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty (deposit accession number PTA-7282).

[0116] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0117] In one example, a significant proportion of STRO-1+ cells are capable of differentiation into at least two different germ lineages. Non-limiting examples of lineages into which STRO-1+ cells may commit include bone precursor cells, hepatocyte progenitors that are multipotent for bile duct epithelial cells and hepatocytes, neural restricted cells that can generate glial cell precursors that progress to oligodendrocytes and astrocytes, neuronal precursors that progress to neurons, cardiac muscle and cardiac muscle cell precursors, glucose-responsive insulin-secreting pancreatic beta cell lines, etc. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, as well as the following precursor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth and skeletal muscle cells, testicular progenitors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes and oligodendrocytes.

[0118] In one example, mesenchymal precursors or stem cells are obtained from a single donor or multiple donors, and the donor samples or the mesenchymal precursors or stem cells are subsequently pooled and then expanded in culture.

[0119] The mesenchymal precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject. In one example, the mesenchymal precursors or stem cells are expanded in culture and cryopreserved prior to administration to a subject.

[0120] In one example, the present disclosure encompasses mesenchymal precursors or stem cells, as well as their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal precursors or stem cells, as well as extracellular vesicles isolated therefrom. For example, the mesenchymal precursors or stem cells of the present disclosure can be cultured and grown for a period of time and under conditions suitable for secreting the extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0121] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but typically they are less than 200 nm in size. They are released from the releasing cells (e.g., mesenchymal stem cells, STRO-1 + The extract may contain proteins, nucleic acids, lipids, metabolic products, or organelles from a living body (such as a human cell).

[0122] The term "exosomes" as used herein refers to a type of extracellular vesicle, generally about 30 nm to about 150 nm in size, derived from endosomal vesicle compartments that are transported to and released from the plasma membrane of mammalian cells. They contain nucleic acids (e.g., RNA, microRNA), proteins, lipids, and metabolites, and can be secreted from one cell, taken up by other cells, and function in cell-cell communication by carrying cargo.

[0123] Cell culture growth In one example, the mesenchymal precursors or stem cells are cultured and expanded. "Cultured and expanded" mesenchymal precursors or stem cells are distinct from freshly isolated cells in that they have been cultured and subcultured (i.e., recultured) in cell culture medium. In one example, the cultured and expanded mesenchymal precursors or stem cells are cultured and expanded for about 4-10 passages. In one example, the mesenchymal precursors or stem cells are cultured and expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5-10 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5-8 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5-7 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for more than 10 passages. In another example, the mesenchymal precursors or stem cells can be culture-expanded for more than seven passages. In these examples, the stem cells may be culture-expanded before being cryopreserved, providing an intermediate cryopreserved MLPSC population. In one example, the composition of the present disclosure is prepared from an intermediate cryopreserved MLPSC population. For example, the intermediate cryopreserved MLPSC population may be further culture-expanded before administration, as further discussed below. Thus, in one example, the mesenchymal precursors or stem cells are culture-expanded and cryopreserved. In one embodiment of these examples, the mesenchymal precursors or stem cells can be obtained from a single donor, or multiple donors, and the donor samples or mesenchymal precursors or stem cells are then pooled and then culture-expanded. In one example, the culture-expansion process includes: i. expanding the number of viable cells by serial expansion to provide a preparation of at least about 1 billion viable cells, the serial expansion comprising establishing a primary culture of isolated mesenchymal precursor or stem cells and then serially establishing a first non-primary (P1) culture of isolated mesenchymal precursor or stem cells from the previous culture; -ii. expanding the P1 culture of isolated mesenchymal precursors or stem cells into a second, non-primary (P2) culture of mesenchymal precursors or stem cells by passaging; -iii. Preparing and cryopreserving an in-process intermediate mesenchymal precursor or stem cell preparation obtained from a P2 culture of mesenchymal precursor or stem cells; and -iv. thawing the cryopreserved in-process intermediate mesenchymal precursor or stem cell preparation and expanding the in-process intermediate mesenchymal precursor or stem cell preparation by sub-propagation.

[0124] In one example, the expanded mesenchymal precursor or stem cell preparation has an antigenic and activity profile that includes: i. less than about 0.75% CD45+ cells; - ii. at least about 95% CD105+ cells; - iii. at least about 95% CD166+ cells.

[0125] In one example, the expanded mesenchymal precursor or stem cell preparation is capable of inhibiting IL2Rα expression by CD3 / CD28-activated PBMCs by at least about 30% compared to a control.

[0126] In one example, the cultured expanded mesenchymal precursors or stem cells are cultured and expanded for about 4-10 passages, where the mesenchymal precursors or stem cells are cryopreserved after at least 2 or 3 passages before being further cultured and expanded. In one example, the mesenchymal precursors or stem cells are cultured and expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages, cryopreserved, and then further cultured and expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages before being administered or further cryopreserved.

[0127] In one example, the majority of the mesenchymal precursors or stem cells in the compositions of the present disclosure are about the same generation (i.e., they are within about 1 or about 2 or about 3 or about 4 cell doublings of each other). In one example, the average number of cell doublings in the compositions is about 20 to about 25 doublings. In one example, the average number of cell doublings in the compositions is about 9 to about 13 (e.g., about 11 or about 11.2) doublings resulting from the primary culture, plus about 1, about 2, about 3, or about 4 doublings per passage (e.g., about 2.5 doublings per passage). Exemplary average cell doublings in the compositions are any of about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5, and about 36 when produced by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, and about 10 passages, respectively.

[0128] The process of mesenchymal precursor or stem cell isolation and ex vivo expansion can be carried out using any equipment and cell handling methods known in the art. Various culture expansion embodiments of the present disclosure use steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing steps. Any step that manipulates cells can damage them. Although mesenchymal precursor or stem cells can generally tolerate some damage during preparation, it is preferable to manipulate cells by handling procedures and / or equipment that properly perform a given step(s) while minimizing damage to the cells.

[0129] In one example, the mesenchymal precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a spun membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, for example as described in US 6,251,295, which is incorporated herein by reference.

[0130] In one example, the mesenchymal precursor or stem cell composition according to the present disclosure is 95% homogeneous with respect to being CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo expansion, i.e., multiple population doublings. In one example, the composition comprises at least one therapeutic dose of mesenchymal precursor or stem cells, the mesenchymal precursor or stem cells comprising less than about 1.25% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD166+ cells. In one example, this homogeneity persists after cryopreservation and thawing, and the cells also generally have a viability of about 70% or more.

[0131] In one example, a composition of the present disclosure includes mesenchymal precursors or stem cells expressing substantial levels of TNF-R1, e.g., greater than 13 pg of TNF-R1 per million mesenchymal precursors or stem cells. In one example, this phenotype is stable through ex vivo expansion and cryopreservation. In one example, expression of levels of TNF-R1 in the range of about 13 to about 179 pg (e.g., about 13 pg to about 44 pg) per million mesenchymal precursors or stem cells is associated with desirable therapeutic potential that persists through ex vivo expansion and cryopreservation.

[0132] In one example, the cultured expanded mesenchymal precursor or stem cells express tumor necrosis factor receptor 1 (TNF-R1) in an amount of at least 110 pg / ml. For example, the mesenchymal precursor or stem cells can express TNF-R1 in an amount of at least 150 pg / ml, or at least 200 pg / ml, or at least 250 pg / ml, or at least 300 pg / ml, or at least 320 pg / ml, or at least 330 pg / ml, or at least 340 pg / ml, or at least 350 pg / ml.

[0133] In one example, the mesenchymal precursor or stem cell has an endothelial cell lineage that is at least 13 pg / 10 6 For example, mesenchymal precursor or stem cells express TNF-R1 at levels of at least 15 pg / 10 6 cells or at least 20 pg / 10 6cells or at least 25 pg / 10 6 cells or at least 30 pg / 10 6 cells or at least 35 pg / 10 6 cells or at least 40 pg / 10 6 cells or at least 45 pg / 10 6 cells or at least 50 pg / 10 6 express TNF-R1 in amounts of 100 cells.

[0134] In another example, the mesenchymal precursors or stem cells disclosed herein inhibit IL-2Rα expression on T cells. In one example, the mesenchymal precursors or stem cells can inhibit IL-2Rα expression by at least about 30%, alternatively at least about 35%, alternatively at least about 40%, alternatively at least about 45%, alternatively at least about 50%, alternatively at least about 55%, alternatively at least about 60%.

[0135] In one example, a composition of the present disclosure comprises at least one therapeutic dose of mesenchymal precursors or stem cells, which can include, for example, at least about 100 million cells or about 125 million cells.

[0136] Cellular modification In one example, the mesenchymal precursor or stem cells of the present disclosure can be modified in such a way that lysis of the cells is inhibited upon administration. Alteration of the antigen can induce immunological unresponsiveness or tolerance, thereby preventing induction of the effector phase of the immune response (e.g., cytotoxic T cell generation, antibody production, etc.) that ultimately leads to rejection of foreign cells in a normal immune response. Antigens that can be modified to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

[0137] Mesenchymal precursors or stem cells may also be genetically modified to express proteins important for the differentiation and / or maintenance of striated skeletal muscle cells. Exemplary proteins include growth factors (TGF-β, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factors (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), members of the neuregulin family (e.g., neuregulin 1, 2, and 3), and homeobox genes (e.g., Csx, Tinman, and NKx families).

[0138] The mesenchymal precursors or stem cells of the present disclosure can also be modified to carry or express an antiviral or thrombolytic agent. In one example, the agent is an antiviral drug. In one example, the agent is an anti-influenza drug. In one example, the agent is anti-SARS-CoV (e.g., SARS-Cov2). An exemplary agent is Remdesivir. In one example, the agent is a thrombolytic drug. Examples of thrombolytic agents include Eminase (anistreplase), Retavase (reteplase), and Streptase (streptokinase, fungal kinase). In one example, the thrombolytic agent is heparin.

[0139] The mesenchymal precursors or stem cells of the present disclosure can be modified to carry antiviral or thrombolytic agents by culturing the cells with the agent for a time and under conditions sufficient to allow the agent to be absorbed by the cells. In one example, the antiviral or thrombolytic agent is added to the culture medium of the mesenchymal precursors or stem cells disclosed herein. For example, the mesenchymal precursors or stem cells disclosed herein can be cultured and grown in a culture medium that includes the antiviral or thrombolytic agent.

[0140] In another example, the antiviral or thrombolytic agent is a peptide. In one example, mesenchymal precursors or stem cells are genetically modified to express an antiviral or thrombolytic peptide or a nucleic acid encoding the same. In one example, mesenchymal precursors or stem cells are modified in vitro through contact with a viral vector. For example, the virus can be added to the cell culture medium. Non-viral methods of genetic modification can also be used. Examples include the application of integrase or transposase technology, liposome or protein transduction domain mediated delivery, and plasmid transfer and targeted gene integration using physical methods such as electroporation.

[0141] The efficiency of genetic modification is rarely 100%, and it is usually desirable to enrich for the population of successfully modified cells. In one example, modified cells can be enriched by taking advantage of functional characteristics of the new genotype. One exemplary method of enriching modified cells is positive selection using a selectable or screenable marker gene. A "marker gene" refers to a gene that confers a distinct phenotype to cells expressing the marker gene, such that such transformed cells can be distinguished from cells that do not possess the marker. A selectable marker gene confers a trait that can be "selected" based on resistance to a selective drug (e.g., an antibiotic). A screenable marker gene (or reporter gene) confers a trait that can be identified through observation or testing, i.e., by "screening" (e.g., β-glucuronidase, luciferase, GFP, or other enzymatic activity not present in non-transformed cells). In one example, genetically modified mesenchymal precursors or stem cells are selected based on resistance to drugs such as neomycin, or colorimetric selection based on expression of lacZ.

[0142] Compositions of the Disclosure In one example of the present disclosure, the mesenchymal precursors or stem cells and / or their progeny and / or soluble factors derived therefrom are administered in the form of a composition.In one example, such a composition comprises a pharma-ceutically acceptable carrier and / or excipient.Thus, in one example, the composition of the present disclosure can comprise culture-expanded mesenchymal precursors or stem cells.

[0143] The terms "carrier" and "excipient" refer to compositions of matter conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce any undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other components within the carrier. In one example, a carrier causes no significant local or systemic adverse effects in a recipient at the dosages and concentrations used therapeutically.

[0144] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, hyaluronan and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0145] In another example, the carrier is, for example, a media composition that the cells are grown or suspended in. For example, such a media composition does not induce any adverse effects in the subject to which it is administered.

[0146] Exemplary carriers and excipients do not adversely affect the viability of the cells and / or the ability of the cells to reduce, prevent, or delay metabolic syndrome and / or obesity.

[0147] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at a suitable pH, thereby exerting biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient since it minimizes cell and factor interaction, allowing for rapid release of cells and factors; in such cases, the compositions of the present disclosure can be prepared as a liquid for direct application, for example, by injection, to the bloodstream or to tissues or areas surrounding or adjacent to the tissue.

[0148] Mesenchymal precursor or stem cells and / or their progeny and / or soluble factors derived therefrom may also be incorporated or embedded into scaffolds that are recipient-compatible and degrade into products that are not harmful to the recipient. These scaffolds provide support and protection to cells that are transplanted into the recipient subject. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds.

[0149] A variety of different scaffolds can be successfully used in the practice of the present disclosure. Exemplary scaffolds include, but are not limited to, biologically degradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Synthetic materials suitable for cell transplantation scaffolds should be able to support a wide range of cell growth and cell functions. Such scaffolds may also be resorbable. Suitable scaffolds include polyglycolic acid scaffolds (e.g., as described in Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima, et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991), or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acids.

[0150] In another example, mesenchymal precursor or stem cells and / or their progeny and / or soluble factors derived therefrom may be administered in a gel scaffold (eg, Gelfoam from the Upjohn Company).

[0151] The compositions disclosed herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the compositions disclosed herein immediately or shortly before administration, or may be co-cultured for a period of time before administration.

[0152] In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of mesenchymal precursor or stem cells, and / or their progeny, and / or soluble factors derived therefrom. For example, the composition may comprise about 1×10 5 Stem cells ~ approx. 1×10 9 stem cells, or approximately 1.25 x 10 3 Stem cells ~ approx. 1.25×10 7 Stem cells / kg (80 kg subject). In one example, the composition contains 2×10 6 The exact amount of cells administered will depend on a variety of factors, including the age, weight, and sex of the subject, as well as the extent and severity of the disorder being treated.

[0153] In one example, 50 x 10 6 ~200×10 7 In another example, 60×10 cells are administered. 6 ~200×10 6 cells or 75 x 10 6 ~150×10 6 In one example, 75 × 10 cells are administered. 6 In another example, 150×10 cells are administered. 6 cells are administered.

[0154] In one example, the composition is 5.00 x 10 6 In another example, the composition comprises more than 5.50×10 viable cells / mL. 6 In another example, the composition comprises more than 6.00×10 viable cells / mL. 6In another example, the composition comprises more than 6.50×10 viable cells / mL. 6 In another example, the composition comprises more than 6.68×10 viable cells / mL. 6 Contains > 10 viable cells / mL.

[0155] In one example, the method of the disclosure includes administering a total dose of 600 million cells. For example, a subject treated according to the disclosure can receive multiple doses of the above reference composition, so long as the total dose of cells does not exceed 600 million cells. For example, a subject can receive three doses of 200 million cells. In one example, the total dose of cells is 500 million cells. In one example, the total dose of cells is 400 million cells. For example, a subject can receive four doses of 100 million cells. In one example, a subject receives one dose of 100 million cells at baseline, followed by three doses of 100 million cells administered once a month for three months. In one example, the dose is 2×10 6 In one example, the dose is 2×10 6 cells / kg and subjects receive two or three doses. In one example, the dose is 2×10 6 cells / kg and subjects receive more than 3 doses.

[0156] In one example, the mesenchymal precursors or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0157] The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal precursors or stem cells can be performed using slow cooling methods or "fast" freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotype, cell surface markers, and growth rate of the cryopreserved cells compared to unfrozen cells.

[0158] The cryopreserved composition may contain a cryopreservation solution, the pH of which is usually 6.5 to 8, preferably 7.4.

[0159] Cryopreservation solutions may include, for example, a sterile, non-pyrogenic, isotonic solution such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride USP (NaCl), 502 mg of sodium gluconate (C6H 11 It contains 368 mg of sodium acetate trihydrate USP (C2H3NaO2·3H2O), 37 mg of potassium chloride USP (KCl), and 30 mg of magnesium chloride USP (MgCl2·6H2O). It does not contain any antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0160] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0161] To facilitate freezing, cryoprotectants such as dimethyl sulfoxide (DMSO) are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high post-thaw survival rates, and allow transplantation without washing. However, the most commonly used cryoprotectant, DMSO, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative to or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0162] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0163] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled rate freezer.

[0164] The cryopreserved composition may be thawed and administered directly to a subject or may be added to another solution, for example, containing HA. Alternatively, the cryopreserved composition may be thawed and the mesenchymal precursors or stem cells resuspended in an alternative carrier prior to administration.

[0165] In one example, a cell composition of the present disclosure can include Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). For example, a composition of the present disclosure can include a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0166] In one example, the compositions described herein may be administered as a single dose.

[0167] In some examples, the compositions described herein may be administered in multiple doses, such as at least two, at least three, or at least four doses. In other examples, the compositions described herein may be administered in at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses.

[0168] In one example, the mesenchymal precursors or stem cells may be cultured and expanded prior to administration to a subject. Various methods of cell culture are known in the art. In one example, the mesenchymal precursors or stem cells are cultured and expanded for about 4 to 10 passages. In one example, the mesenchymal precursors or stem cells are cultured and expanded for at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. In one example, the mesenchymal precursors or stem cells are cultured and expanded for at least 5 passages. In these examples, the stem cells may be cultured and expanded prior to cryopreservation.

[0169] In one example, the mesenchymal precursors or stem cells are cultured and expanded in serum-free medium prior to administration.

[0170] In some instances, the cells are contained within a chamber that does not allow the cells to exit the circulation of the subject, but does allow factors secreted by the cells to enter the circulation. In this manner, soluble factors can be administered to a subject by allowing the cells to secrete the factors into the circulation of the subject. Such chambers can similarly be implanted at a site in a subject to increase the local level of soluble factors.

[0171] In one example, the mesenchymal precursors or stem cells may be administered systemically. In one example, the mesenchymal precursors or stem cells may be administered to the airways of the subject. In one example, the mesenchymal precursors or stem cells may be administered to the lung(s) of the subject. In another example, the compositions of the present disclosure are administered intravenously. In another example, the compositions are administered intravenously and to the airways of the subject.

[0172] In one example, the mesenchymal precursors or stem cells are administered once a week. For example, the mesenchymal precursors or stem cells can be administered once a week for every two weeks. In another example, the mesenchymal precursors or stem cells can be administered twice a week. In one example, the mesenchymal precursors or stem cells can be administered once a month. In one example, two doses of mesenchymal precursors or stem cells are administered once a week for two weeks. In another example, two doses of mesenchymal precursors or stem cells are administered once a week for every two weeks. In another example, four doses of mesenchymal precursors or stem cells are administered for two weeks before a subsequent dose is administered monthly. In one example, two doses of mesenchymal precursors or stem cells can be administered once a week for every two weeks before a subsequent dose is administered monthly. In one example, four doses are administered monthly.

[0173] In one example, the composition of the present disclosure comprises a "clinically proven effective" amount of MLPSCs. In one example, the composition of the present disclosure comprises a "clinically proven effective" amount of MSCs.

[0174] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0175] The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. EXAMPLES

[0176] Ex vivo expanded adult allogeneic bone marrow-derived mesenchymal stem cells (MSCs) for the treatment of acute respiratory distress syndrome (ARDS) composition The composition is comprised of culture-expanded mesenchymal stem cells (ceMSCs) isolated from the bone marrow of healthy adult donors. The final composition contains ceMSCs formulated in Plasma-Lyte A, dimethyl sulfoxide (DMSO) and human serum albumin (HSA).

[0177] the purpose To determine: -Safety -Improved survival rates.

[0178] subject Patients characterized as having moderate COVID-19-associated ARDS received mesenchymal stem cells (intravenous, 2 million cells per kg) or control therapy. 125 patients were <65 years old (i.e., the intention to treat (ITT) population, Table 1). 58 of the ITT population received cell therapy and 67 received control therapy. Some ITT patients were identified as having mild ARDS and / or low levels of inflammation, as characterized by circulating CRP <4. These patients were excluded from the per-protocol (PP) patient population. Thus, the PP population was reduced to 89 (from 125), with 38 receiving cell therapy and 51 receiving control therapy. Furthermore, the PP population consistently represents the study population with the most severe disease, as they all had moderate to severe ARDS. [Table 2]

[0179] analysis Cell therapy provided some degree of protection against death in all patients at day 60 (Figure 1).

[0180] However, further analysis of treated patients surprisingly revealed that cell therapy provided significant protection against 60-day mortality in patients under 65 years of age (Figures 2, 3, and 9; A) compared to patients over 65 years of age (Figures 2, 3, and 9; B). Significantly, protection against 60-day mortality in patients under 65 years of age was evident in both intention-to-treat (ITT) patients (38.3% reduction in mortality compared to controls, p=0.0484, Figure 2) and per-protocol (PP) patients (43.3% reduction in mortality compared to controls, p=0.0285, Figure 3). As also shown in Figures 2 and 3, a greater reduction in mortality was observed in PP patients compared to ITT patients. This is an unexpected outcome, as PP patients consistently had more severe disease than ITT patients. These data suggest that subjects with more severe ARDS, such as moderate to severe ARDS, may respond better to treatment with cell therapy.

[0181] A significant protection against death was also observed in patients who were under 60 years of age and who received dexamethasone at baseline, further supporting the rationale for selecting ARDS patients under 65 years of age for treatment with cell therapy, especially if subjects were also receiving corticosteroids (Figure 4: A, ITT patients, 68.9% reduction in death, p=0.0074; B, PP patients, 78.9% reduction in death, p=0.0043).

[0182] Patients under 65 years of age are generally expected to have a higher health status than patients over 65 years of age. Despite their higher health status, these patients have died from ARDS and have a surprisingly improved outcome after treatment compared to patients over 65 years of age. These data support the identification of a population of ARDS patients who will respond well to cell therapy, especially considering the protection offered against mortality in these patients.

[0183] Analysis of the control population revealed that administration of dexamethasone at baseline did not reduce mortality in control patients (Figure 5: A: all control ITT patients <65 years old, p=0.554; B: all control ITT patients ≥65 years old, p=0.815). Surprisingly, this lack of protection against death at 60 days was rectified in patients treated with cell therapy and dexamethasone (Figure 4), and more than additive protection against death was observed in patients treated with cell therapy and dexamethasone (Figures 6, 7, and 8). Significantly, the observed synergy between cell therapy and dexamethasone was prominent in both ITT patients (p=0.0669) and PP patients (p=0.0363). The combination of cell therapy with dexamethasone was also synergistic in increasing 60-day ventilator-free survival days in an exploratory analysis of patients <65 years old (Figure 10).

[0184] Protection against death in patients under 65 years of age is summarized in Figure 6. No protection was provided to control patients or patients who received dexamethasone alone. Treatment with cell therapy conferred some protection against death, which was significantly increased by treatment with cell therapy and dexamethasone (see also Figure 4). Both clinical and respiratory functional improvements were observed after combination of cell therapy with dexamethasone (Figures 7 and 8). This combination also increased the number of ventilator-free days that patients remained alive (Figure 10).

[0185] These data provide a compelling rationale for selecting patients with ARDS under the age of 65 for treatment with cell therapy, and in particular for treatment with cell therapy and corticosteroids such as dexamethasone. These data also suggest that by selecting ARDS patients under the age of 65 who are taking corticosteroids and administering cell therapy to these patients, a significant improvement in treatment outcomes can be achieved in ARDS patients.

[0186] Biomarker analysis Inflammatory biomarker levels were monitored at baseline and within 3–5 days of cell therapy (2 million (2 × 106 After treatment with 107 treated patients and 106 controls (treated <65 n=55, control <65 n=65, treated >65 n=52, control >65 n=41), cell therapy significantly reduced CRP levels on days 3, 7, and 14 (FIG. 11A) and ferritin levels on days 7 and 14 (FIG. 11B) in patients <65 years of age. Cell therapy prevented a significant increase in D-dimer levels in all treated patients (FIG. 11C).

[0187] Comparing inflammatory biomarker levels between treated subjects <65 years old and >65 years old provided a unique opportunity to identify measurable criteria for selecting patients for treatment. Surprisingly, as shown in Figures 12 and 13, age >65 years seems to be associated with increased inflammatory activity at baseline, regardless of whether the patient was using corticosteroids or not. Indeed, an analysis of all patients using corticosteroids at baseline showed >5-fold higher inflammatory activity (cytokines / chemokines) in patients >65 years old compared to patients <65 years old.

[0188] FIG. 12 shows that patients over 65 years of age had higher baseline levels of inflammatory cytokines / chemokines than patients <65 years of age, in particular: (i) CCR2-binding chemokines (including CXCL10 / IP10 and CXCL9) and CXCR3-binding chemokines (including CCL2, CCL3, and CCL7 / MCP3). This group of chemokines is shown to increase neutrophil and macrophage influx into the lung. (ii) IL-6 and IL-8, which indicate increased macrophage inflammation and enhanced neutrophil migration to the lungs. (ii) CCL19 and IL-2, which indicate T cell activation / proliferation and apoptotic death. This indicates that the

[0189] Other inflammatory biomarkers increased in patients with >65 included TGF-alpha, TNF, CXCL8, G-CSF / CSF-3, and IL17C (Figures 12 and 13).

[0190] Patients <65 years of age expressed the same inflammatory pathways as patients >65 years of age, albeit at lower baseline levels. In patients <65 years of age, cell therapy significantly reduced inflammatory markers associated with COVID-19 ARDS severity in patients <65 years of age, specifically: - chemokines that bind to CCR2 and CXCR3, likely resulting in a reduction in neutrophil and macrophage influx into the lungs; -IL-6 / IL-8 / TNF / IL-18, showing a decrease in IL-18-driven inflammasome and a decrease in macrophage activation / neutrophil homing to the lungs; - Decreased CCL19, and reduced IL-4 / IL-13 / GM-CSF, indicating reduced T cell influx and activation.

[0191] In summary: - In treated patients aged <65 years, inflammatory pathways were downregulated (Figures 12 and 13), which corresponded to improved prognosis (Figures 2-4) and sustained improvement in respiratory function (Figure 8); Improved respiratory function was observed in treated patients >65 years of age on day -7, but was not sustained (Figure 8); subsequent biomarker analysis revealed that these patients had higher baseline levels of inflammation (Figures 12 and 13); - Biomarker analysis revealed that similar inflammatory pathways were involved in patients with ARDS, regardless of age (<65 vs. >65 years).

[0192] (safety) No infusion-related adverse events were observed.

[0193] Those skilled in the art will appreciate that many variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit and scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0194] All publications discussed above are incorporated herein in their entirety.

[0195] This application claims priority from AU2021901214, filed April 23, 2021, AU2021902180, filed July 15, 2021, AU2022900260, filed February 9, 2022, and AU2022900372, filed February 18, 2022, the disclosures of which are incorporated herein by reference.

[0196] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and should not be construed as an admission that any or all of those matters formed part of the prior art base or were general general knowledge in the fields relevant to the present invention prior to the priority date of each claim of this application.

Claims

1. A composition for treating or preventing acute respiratory distress syndrome (ARDS) in a human subject in need thereof, comprising a population of mesenchymal precursor or stem cells (MLPSCs), wherein the subject is under 65 years of age and is taking corticosteroids.

2. The composition of claim 1 , wherein the ARDS is moderate or severe.

3. 2. The composition of claim 1, wherein the subject is under 60 years old, or the subject is between 18 and 65 years old, or the subject is between 18 and 60 years old.

4. The composition of claim 1 , wherein the corticosteroid is dexamethasone.

5. 2. The composition of claim 1, wherein the ARDS is caused by a viral infection, and the virus is a rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus.

6. 6. The composition of claim 5, wherein the coronavirus is Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), or COVID-19.

7. The composition of claim 1 , wherein the ARDS is caused by thrombosis, venous thrombosis, arterial thrombosis, or pulmonary embolism.

8. 10. The composition of claim 1, wherein the treated subject's risk of death is reduced after treatment, or the treated subject's risk of death is reduced by 30-60%, or the treated subject has an improved 60-day survival rate.

9. The composition of claim 1 , wherein the MLPSCs are cryopreserved and thawed, or the MLPSCs are culture expanded from an intermediate cryopreserved MLPSC population.

10. The composition of claim 1 , wherein the MLPSCs are mesenchymal stem cells (MSCs).

11. The composition of claim 1 , wherein the MLPSCs are allogeneic or the MLPSCs are modified to carry or express an antiviral or thrombolytic agent.

12. 1 x 10 per dose ７ ~2×10 ８ 10. The composition of claim 1, comprising 1 x 108 cells per dose, or 1 x 108 cells per dose, or two doses each comprising 2 x 106 cells per kilogram of body weight.

13. The composition of claim 12 , wherein the subject receives two doses.

14. 14. The composition of claim 13, wherein the subject receives a second dose within seven days of receiving a first dose, or the second dose is administered four days after the first dose.

15. 2. The composition of claim 1, further comprising a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, the HSA solution comprising 5% HSA and 15% buffer, and the composition comprising greater than 6.68 x 10 viable cells / mL.

16. The composition of claim 1 , wherein the subject is on a ventilator.

17. 17. The composition of claim 16, wherein the subject is weaned off the ventilator following treatment, or the subject is weaned off the ventilator within 60 days of treatment.

18. The treatment reduces the level of at least one inflammatory biomarker compared to baseline, and the at least one inflammatory biomarker is (a) a reduction in neutrophil and macrophage influx into the lungs; (b) a decrease in inflammasomes; (c) reduced macrophage activation and neutrophil migration to the lungs; (d) a decrease in T cell influx and activation, or (e) the composition of claim 1, exhibiting a reduction in circulating biomarkers of macrophage and neutrophil inflammation.

19. The inflammatory biomarker is selected from the group consisting of: - CXCR3-binding chemokines, preferably CXCL10 and / or CXCL9, - CCR2-binding chemokines, preferably CCL2, CCL3, and / or CCL7, - IL-6, - IL-8, - TNF, - IL-18, - CCL19, - IL-4, - IL-13, - GM-CSF, - CRP, or 20. The composition of claim 18, wherein the protein is one or more of ferritin.

20. 2. The composition of claim 1, wherein the treatment reduces CRP and / or ferritin levels within 3-14 days of administering MLPSCs, or respiratory function as defined by the Berlin Criteria is improved on day 14 and / or day 21.

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