Methods and compositions for treating lung diseases

Abstract

The present invention provides compositions comprising mesenchymal stem cells (MSCs) and / or exosomes derived therefrom, or mesenchymal stem cells that secrete neurotrophic factors (MSC-NTFs) and / or exosomes derived therefrom, and methods for using them in the treatment of severe lung diseases, such as coronavirus-associated acute respiratory distress syndrome (ARDS).

Description

Technical Field

[0001] The present invention relates to methods and compositions for treating lung diseases.

Background Art

[0002] The body's respiratory system includes the nose, sinuses, mouth, pharynx, larynx, trachea, and lungs. Upper respiratory tract infections affect the upper airway parts of the body such as the nose, sinuses, and larynx, and lower respiratory tract infections affect the airway and lungs.

[0003] Types of upper respiratory tract infections include colds (nasal colds), mild influenza, tonsillitis, laryngitis, and sinus infections. Among the symptoms of upper respiratory tract infections, the most common is cough. Lung infections also cause nasal congestion, runny nose, sore throat, sneezing, muscle pain, headache, etc.

[0004] Lower respiratory tract infections are seen in the lungs and respiratory airways. Lower respiratory tract infections are caused by viral infections such as severe influenza and bacterial infections such as tuberculosis. Symptoms of lower respiratory tract infections include severe coughing with mucus (phlegm), shortness of breath, chest tightness, and wheezing when exhaling.

[0005] The COVID-19 pandemic caused by SARS-CoV-2 causes mild, moderate, or severe diseases. Severe clinical symptoms include pneumonia, acute respiratory distress syndrome (ARDS), sepsis, septic shock, etc.

[0006] Although the proportion of affected patients is still undetermined, about one week after onset, a sudden clinical deterioration accompanied by rapidly progressing respiratory failure, multiple organ dysfunction (MOD), and multiple organ failure (MOF) is observed.

[0007] Concerns that COVID-19 may cause severe illness, respiratory failure, and death have become the core of social unrest. Acute respiratory distress syndrome (ARDS) caused by COVID-19 is associated with a mortality rate of over 50%. However, there is currently no effective radical treatment strategy for ARDS, which is a type of respiratory failure accompanied by extensive inflammation and abnormal cytokine production that can be confirmed by bronchoalveolar lavage (BAL). In addition, despite recent advances and intensive care, ARDS is accompanied by severe systemic inflammation and multiple organ failure, and the complex impact on existing medical resources is unacceptably large. The viral load of COVID-19 has been demonstrated to correlate with lung function, outcome, and inflammatory biomarkers, suggesting a synergistic effect between a treatment that inhibits viral growth and a treatment that inhibits lung inflammatory diseases. It is clear that there is a desperate need for a treatment that can minimize the impact of COVID-19 on ARDS, sepsis, and multiple organ failure.

[0008] Furthermore, improved treatment methods and treatment strategies are needed to address the ongoing and future COVID-19 pandemics.

Summary of the Invention

Means for Solving the Problems

[0009] In one aspect, the present invention is a method for treating a viral lung infection or its symptoms in a patient in need of treatment, comprising: (a) a plurality of multipotent mesenchymal stem cells (MSCs) or a plurality of mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs); (b) EXO-MSC defined as a plurality of small extracellular vesicles (sEVs) derived from multipotent mesenchymal stem cells (MSCs), or mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs) (NurOwn (Registered Trademark in Foreign Countries))Provided is a method having a step of administering to a patient, using a therapeutically effective regimen, a pharmaceutical composition comprising an active agent selected from the group consisting of EXO-MSC-NTF defined as a plurality of small extracellular vesicles (sEV) derived from [[ID=]], and (c) a combination of a pluripotent mesenchymal stem cell (MSC) or a neurotrophic factor-secreting mesenchymal stem cell (MSC-NTF) and EXO-MSC or EXO-NSC-NTF.

[0010] In some embodiments, the therapeutically effective regimen comprises a single administration of the active agent. In some embodiments, the therapeutically effective regimen comprises repeated administrations of the active agent.

[0011] In some embodiments, the active agent is a pluripotent mesenchymal stem cell (MSC). In some embodiments, the active agent is a combination of a pluripotent mesenchymal stem cell (MSC) and EXO-MSC.

[0012] In some embodiments, the active agent is a neurotrophic factor-secreting mesenchymal stem cell (MSC-NTF).

[0013] In some embodiments, the pharmaceutical composition comprises from about 5×10 6 to about 300×10 6 pluripotent mesenchymal stem cells (MSCs).

[0014] In some embodiments, the pharmaceutical composition comprises from about 15×10 6 to about 100×10 6 pluripotent mesenchymal stem cells (MSCs).

[0015] In some embodiments, the pharmaceutical composition comprises from about 15×10 6 to about 20×10 6 pluripotent mesenchymal stem cells (MSCs).

[0016] In some embodiments, the pharmaceutical composition comprises from about 80×10 6 to about 100×10 6 pluripotent mesenchymal stem cells (MSCs).

[0017] In some embodiments, the active agent is EXO-MSC.

[0018] In some embodiments, the active agent is EXO-MSC-NTF.

[0019] In some embodiments, the pharmaceutical composition comprises from about 10 9 to about 10 12 EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises from about 10 10 to about 10 12 EXO-MSCs or EXO-MSC-NTFs.

[0020] In some embodiments, the pharmaceutical composition comprises from about 3×10 10 to about 3×10 11 EXO-MSCs or EXO-MSC-NTFs.

[0021] In some embodiments, the pharmaceutical composition comprises about 10 11 EXO-MSCs or EXO-MSC-NTFs.

[0022] In some embodiments, the total amount of active agent administered to a patient is (a) from about 75×10 6 to about 500×10 6 pluripotent mesenchymal stem cells (MSCs), (b) about 5×10 11 EXO-MSCs or EXO-MSC-NTFs, and (c) a combination of from about 75×10 6 to about 500×10 6 MSCs and about 5×10 11 EXO-MSCs or EXO-MSC-NTFs, selected from the group consisting of.

[0023] In some embodiments, the pluripotent mesenchymal stem cells (MSCs) include bone marrow-derived MSCs (BM-MSCs).

[0024] In some embodiments, the therapeutically effective regimen includes a single administration of the active agent. In some embodiments, the therapeutically effective regimen includes repeated administrations of the active agent.

[0025] In some embodiments, the therapeutically effective regimen includes repeated administrations of the active agent on different days.

[0026] In some embodiments, repeated administration includes administering on at least 5 different days.

[0027] In some embodiments, repeated administration includes administering daily.

[0028] In some embodiments, repeated administration includes administering every other day.

[0029] In some embodiments, the pharmaceutical composition further comprises an excipient.

[0030] In some embodiments, the excipient is PlasmaLyte A.

[0031] In some embodiments, the excipient is DMEM.

[0032] In some embodiments, the excipient is CryoStor® CS10 cryopreservation medium.

[0033] In some embodiments, the volume of the pharmaceutical composition is from about 100 mL to about 120 mL.

[0034] In some embodiments, the methods of the present disclosure include systemic administration of the pharmaceutical composition.

[0035] In some embodiments, the methods of the present disclosure include intravenous administration of the pharmaceutical composition.

[0036] In some embodiments, the methods of the present disclosure include intranasal administration of the pharmaceutical composition.

[0037] In some embodiments, the methods of the present disclosure include inhalation administration of a pharmaceutical composition.

[0038] In some embodiments, the methods of the present disclosure include intratracheal administration of a pharmaceutical composition.

[0039] In some embodiments, the methods of the present disclosure include direct injection of a pharmaceutical composition.

[0040] In some embodiments, the methods of the present disclosure include administration of a pharmaceutical composition by inhalation.

[0041] In some embodiments, the symptoms are selected from the group consisting of pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, fever, dry cough, fatigue, sputum production, loss of smell, shortness of breath, muscle pain, joint pain, sore throat, headache, chills, nausea, vomiting, nasal congestion, and diarrhea.

[0042] In some embodiments, the symptom is pneumonia.

[0043] In some embodiments, the symptom is acute respiratory distress syndrome (ARDS).

[0044] In some embodiments, the viral lung infection is selected from the group consisting of coronavirus infection, severe acute respiratory syndrome (SARS) infection, Middle East respiratory syndrome (MERS) infection, influenza virus infection, Ebola virus infection, rabies virus infection, West Nile virus infection, dengue virus infection, respiratory syncytial virus (RSV) infection, and Zika virus infection.

[0045] In some embodiments, the viral lung infection is a coronavirus infection. In some embodiments, the active agent is selected from the group consisting of: (a) a plurality of mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs), (b) EXO-MSC-NTFs defined as a plurality of small extracellular vesicles (sEVs) derived from mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs), and (c) a combination of mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs) and EXO-NSC-NTFs.

[0046] Other aspects and features of the present disclosure will become apparent to those skilled in the art by reading the following description of specific embodiments in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0047]

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DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides methods, compositions, and treatment regimens for treating various human diseases generally known as "viral lung infections" or "respiratory viral infections".

[0049] According to the principles of the present invention, in order to maximize the therapeutic effect of a therapeutic agent and minimize the inconvenience and risks to the patient being treated, the therapeutic agent is administered to the patient at a predetermined dose and according to a predetermined treatment regimen.

[0050] As will be understood by those skilled in the art, different therapeutic results will be obtained if different therapeutic agents are administered at different doses and by different administration methods.

[0051] In one aspect, the present invention provides a method for treating viral lung infections or their symptoms in a patient in need of treatment, comprising administering to the patient a pharmaceutical composition comprising an active agent selected from the group consisting of: (a) a plurality of pluripotent mesenchymal stem cells (MSCs) or a plurality of mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs); (b) EXO-MSC defined as a plurality of small extracellular vesicles (sEVs) derived from pluripotent mesenchymal stem cells (MSCs), or EXO-MSC-NTF defined as a plurality of small extracellular vesicles (sEVs) derived from mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs); and (c) a combination of pluripotent mesenchymal stem cells (MSCs) or mesenchymal stem cells secreting neurotrophic factors (MSC-NTFs) with EXO-MSC or EXO-MSC-NTF, using a therapeutically effective regimen. (NurOwn (Registered Trademark in Foreign Countries))

[0052] In some embodiments, the therapeutically effective regimen comprises a single administration of the active agent. In some embodiments, the therapeutically effective regimen comprises repeated administrations of the active agent. In some embodiments, the therapeutically effective regimen comprises multiple dosing events of the active agent. In some embodiments, the therapeutically effective regimen comprises multiple dosing events of the same active agent. In some embodiments, the therapeutically effective regimen comprises multiple dosing events of different active agents. In some embodiments, the therapeutically effective regimen comprises single dosing events of each of different active agents. In some embodiments, the therapeutically effective regimen comprises administration of two different active agents. In some embodiments, the therapeutically effective regimen comprises administration of three different active agents.

[0053] ​In some embodiments, the active agent is a multipotent mesenchymal stem cell (MSC). In some embodiments, the MSC is administered to the patient at least twice. In some embodiments, the MSC is administered to the patient at least three times. In some embodiments, the MSC is administered to the patient at least four times. In some embodiments, the MSC is administered to the patient at least five times. In some embodiments, the MSC is administered to the patient twice. In some embodiments, the MSC is administered to the patient three times. In some embodiments, the MSC is administered to the patient four times. In some embodiments, the MSC is administered to the patient five times. In some embodiments, the MSC is administered to the patient fewer than two times. In some embodiments, the MSC is administered to the patient fewer than three times. In some embodiments, the MSC is administered to the patient fewer than four times. In some embodiments, the MSC is administered to the patient fewer than five times. In some embodiments, the MSC is administered to the patient one to five times. In some embodiments, the MSC is administered to the patient two to five times. In some embodiments, the MSC is administered to the patient three to five times. In some embodiments, the MSC is administered to the patient four to five times.

[0054] In some embodiments, the MSC is administered on the 1st, 3rd, and 5th days. In some embodiments, the MSC is administered on the 1st, 3rd, 5th, 7th, and 9th days.

[0055] In some embodiments, the active agent is a combination of MSC and EXO-MSC. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient at least twice. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient at least three times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient at least four times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient at least five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient twice. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient three times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient four times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient less than or equal to two times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient less than or equal to three times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient less than or equal to four times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient less than or equal to five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient one to five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient two to five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient three to five times. In some embodiments, the combination of MSC and EXO-MSC is administered to the patient four to five times.

[0056] In some embodiments, the combination of MSC and EXO-MSC is administered on the 1st, 3rd, and 5th days. In some embodiments, the combination of MSC and EXO-MSC is administered on the 1st, 3rd, 5th, 7th, and 9th days.

[0057] In some embodiments, the pharmaceutical composition is about 1×105 ~ about 1000×10 7 contains MSCs. In some embodiments, the pharmaceutical composition is about 5×10 5 ~ about 300×10 7 consisting of MSCs. In some embodiments, the pharmaceutical composition is about 1×10 6 ~ about 1000×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 5×10 6 ~ about 300×10 6 contains MSCs.

[0058] In some embodiments, the pharmaceutical composition is about 5×10 6 ~ about 300×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 1×10 6 ~ about 200x10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 15×10 6 ~ about 100×10 6 contains MSCs.

[0059] In some embodiments, the pharmaceutical composition is about 5×10 6 ~ about 60×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 1×10 6 ~ about 40×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 15×10 6 ~ about 20×10 6 contains MSCs.

[0060] In some embodiments, the pharmaceutical composition is about 20×10 6 ~ about 400×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 40×10 6 ~ about 200×10 6 contains MSCs. In some embodiments, the pharmaceutical composition is about 80×10 6 ~ about 100×10 6 contains MSCs.

[0061] In some embodiments, the active agent is EXO-MSC. In some embodiments, EXO-MSC is administered to the patient at least twice. In some embodiments, EXO-MSC is administered to the patient at least three times. In some embodiments, EXO-MSC is administered to the patient at least four times. In some embodiments, EXO-MSC is administered to the patient at least five times. In some embodiments, EXO-MSC is administered to the patient twice. In some embodiments, EXO-MSC is administered to the patient three times. In some embodiments, EXO-MSC is administered to the patient four times. In some embodiments, EXO-MSC is administered to the patient five times. In some embodiments, EXO-MSC is administered to the patient less than or equal to two times. In some embodiments, EXO-MSC is administered to the patient less than or equal to three times. In some embodiments, EXO-MSC is administered to the patient less than or equal to four times. In some embodiments, EXO-MSC is administered to the patient less than or equal to five times. In some embodiments, EXO-MSC is administered to the patient one to five times. In some embodiments, EXO-MSC is administered to the patient two to five times. In some embodiments, EXO-MSC is administered to the patient three to five times. In some embodiments, EXO-MSC is administered to the patient four to five times.

[0062] In some embodiments, EXO-MSC is administered on the 1st, 3rd, and 5th days. In some embodiments, EXO-MSC is administered on the 1st, 3rd, 5th, 7th, and 9th days.

[0063] In some embodiments, the active agent is EXO-MSC-NTF. In some embodiments, EXO-MSC-NTF is administered to the patient at least twice. In some embodiments, EXO-MSC-NTF is administered to the patient at least three times. In some embodiments, EXO-MSC-NTF is administered to the patient at least four times. In some embodiments, EXO-MSC-NTF is administered to the patient at least five times. In some embodiments, EXO-MSC-NTF is administered to the patient two times. In some embodiments, EXO-MSC-NTF is administered to the patient three times. In some embodiments, EXO-MSC-NTF is administered to the patient four times. In some embodiments, EXO-MSC-NTF is administered to the patient five times. In some embodiments, EXO-MSC-NTF is administered to the patient less than or equal to two times. In some embodiments, EXO-MSC-NTF is administered to the patient less than or equal to three times. In some embodiments, EXO-MSC-NTF is administered to the patient less than or equal to four times. In some embodiments, EXO-MSC-NTF is administered to the patient less than or equal to five times. In some embodiments, EXO-MSC-NTF is administered to the patient one to five times. In some embodiments, EXO-MSC-NTF is administered to the patient two to five times. In some embodiments, EXO-MSC-NTF is administered to the patient three to five times. In some embodiments, EXO-MSC-NTF is administered to the patient four to five times.

[0064] In some embodiments, EXO-MSC-NTF is administered on the first, third, and fifth days. In some embodiments, EXO-MSC-NTF is administered on the first, third, fifth, seventh, and ninth days.

[0065] In some embodiments, the pharmaceutical composition comprises from about 10 9 to about 10 13 EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises from about 3×10 9 to about 3×10 12comprises individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 10 9 to about 10 12 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 10 10 to about 10 12 individual EXO-MSCs or EXO-MSC-NTFs.

[0066] In some embodiments, the pharmaceutical composition comprises about 2×10 9 to about 5×10 11 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 3×10 9 to about 3×10 11 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 5×10 9 to about 2×10 11 individual EXO-MSCs or EXO-MSC-NTFs.

[0067] In some embodiments, the pharmaceutical composition comprises about 2×10 10 to about 5×10 11 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 3×10 10 to about 3×10 11 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 5×10 10 to about 2×10 11 individual EXO-MSCs or EXO-MSC-NTFs.

[0068] In some embodiments, the pharmaceutical composition comprises about 10 11 individual EXO-MSCs or EXO-MSC-NTFs. In some embodiments, the pharmaceutical composition comprises about 0.9×10 11 to about 1.1×10 11 individual EXO-MSCs or EXO-MSC-NTFs.

[0069] In some embodiments, the total amount of active agent administered to the patient is (a) about 75×10 5 to about 500×10 7 MSC, (b) about 5×10 10 to about 5×10 12 EXO-MSC or EXO-MSC-NTF, and (c) about 75×10 5 to about 500×10 7 MSC and about 5×10 9 to about 5×10 12 EXO-MSC or EXO-MSC-NTF, and is selected from the group consisting of combinations thereof.

[0070] In some embodiments, the total amount of active agent administered to the patient is (a) about 75×10 5 to about 500×10 7 MSC, (b) about 5×10 10 to about 5×10 12 EXO-MSC or EXO-MSC-NTF, and (c) about 75×10 5 to about 500×10 7 MSC and about 5×10 10 to about 5×10 12 EXO-MSC or EXO-MSC-NTF, and is selected from the group consisting of combinations thereof.

[0071] In some embodiments, the total amount of active agent administered to the patient is (a) about 15×10 6 to about 250×10 7 MSC, (b) about 1×10 11 to about 25×10 11 EXO-MSC or EXO-MSC-NTF, and (c) about 15×10 6 to about 250×10 7 MSC and about 1×10 11 to about 25×10 11 1 EXO-MSC or EXO-MSC-NTF, and is selected from the group consisting of combinations thereof.

[0072] In some embodiments, the total amount of active agent administered to the patient is (a) about 25×10 6~ about 150×10 7 of MSCs, (b) about 1.5×10 11 ~ about 1.5×10 12 of EXO-MSCs or EXO-MSC-NTFs, and (c) about 25×10 6 ~ about 150×10 7 of MSCs and about 1.5×10 11 ~ about 1.5×10 12 of EXO-MSCs or EXO-MSC-NTFs, and is selected from the group consisting of combinations thereof.

[0073] In some embodiments, the total amount of active agent administered to a patient is (a) about 30×10 6 ~ about 100×10 7 of MSCs, (b) about 2.5×10 11 ~ about 1×10 12 of EXO-MSCs or EXO-MSC-NTFs, and (c) about 30×10 6 ~ about 100×10 7 of MSCs and about 2.5×10 11 ~ about 1×10 12 of EXO-MSCs or EXO-MSC-NTFs, and is selected from the group consisting of combinations thereof.

[0074] In some embodiments, the total amount of active agent administered to a patient is (a) about 75×10 6 ~ about 500×10 6 of MSCs, (b) about 5×10 11 of EXO-MSCs or EXO-MSC-NTFs, and (c) about 75×10 6 ~ about 500×10 6 of MSCs and about 5×10 11 of EXO-MSCs or EXO-MSC-NTFs, and is selected from the group consisting of combinations thereof.

[0075] In some embodiments, the MSCs include bone marrow-derived MSCs (BM-MSCs). In some embodiments, the MSCs consist of BM-MSCs.

[0076] In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent at different months. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent at different weeks. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent at different days. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent at different times of the same day.

[0077] In some embodiments, repeated administration comprises administering on different days for at least 2 days. In some embodiments, repeated administration comprises administering on different days for at least 3 days. In some embodiments, repeated administration comprises administering on different days for at least 4 days. In some embodiments, repeated administration comprises administering on different days for at least 5 days.

[0078] In some embodiments, repeated administration comprises administering daily. In some embodiments, repeated administration comprises administering continuously for at least 2 days. In some embodiments, repeated administration comprises administering continuously for at least 3 days. In some embodiments, repeated administration comprises administering continuously for at least 4 days. In some embodiments, repeated administration comprises administering continuously for at least 5 days.

[0079] In some embodiments, repeated administration comprises administering every other day. In some embodiments, repeated administration comprises administering on the 1st, 3rd, and 5th days. In some embodiments, repeated administration comprises administering on the 1st, 3rd, 5th, 7th, and 9th days.

[0080] In some embodiments, the pharmaceutical composition further comprises an excipient. In some embodiments, the excipient is Plasma-Lyte A.

[0081] In some embodiments, the volume of the pharmaceutical composition is from about 100 mL to about 120 mL. In some embodiments, the volume of the pharmaceutical composition is 104 mL. In some embodiments, the volume of the pharmaceutical composition is 110 mL. In some embodiments, the volume of the pharmaceutical composition is 114 mL.

[0082] In some embodiments, the methods of the present disclosure include systemic administration of the pharmaceutical composition. In some embodiments, the methods of the present disclosure include intravenous administration of the pharmaceutical composition. In some embodiments, the methods of the present disclosure include intratracheal administration of the pharmaceutical composition.

[0083] In some embodiments, the pharmaceutical composition is fresh. In some embodiments, the pharmaceutical composition is not frozen. In some embodiments, the pharmaceutical composition is frozen. In some embodiments, the pharmaceutical composition is thawed after being frozen. In some embodiments, the active agent is fresh. In some embodiments, the active agent is not frozen. In some embodiments, the active agent is frozen. In some embodiments, the active agent is thawed after being frozen.

[0084] In some embodiments, the symptoms are selected from the group consisting of pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, fever, dry cough, fatigue, sputum production, loss of smell, shortness of breath, muscle pain, joint pain, sore throat, headache, chills, nausea, vomiting, nasal congestion, and diarrhea.

[0085] In some embodiments, the symptom is pneumonia. In some embodiments, the symptom is ARDS. In some embodiments, the symptom is multiple organ failure. In some embodiments, the symptom is fever. In some embodiments, the symptom is a dry cough. In some embodiments, the symptom is fatigue. In some embodiments, the symptom is sputum production. In some embodiments, the symptom is anosmia. In some embodiments, the symptom is shortness of breath. In some embodiments, the symptom is myalgia. In some embodiments, the symptom is arthralgia. In some embodiments, the symptom is pharyngitis. In some embodiments, the symptom is headache. In some embodiments, the symptom is chills. In some embodiments, the symptom is nausea. In some embodiments, the symptom is vomiting. In some embodiments, the symptom is nasal congestion. In some embodiments, the symptom is diarrhea.

[0086] In some embodiments, the viral lung infection is selected from the group consisting of coronavirus infection, severe acute respiratory syndrome (SARS) infection, Middle East respiratory syndrome (MERS) infection, influenza virus infection, Ebola virus infection, rabies virus infection, West Nile virus infection, dengue virus infection, respiratory syncytial virus (RSV) infection, and Zika virus infection.

[0087] In some embodiments, the viral lung infection is a coronavirus infection. In some embodiments, the viral lung infection is a severe acute respiratory syndrome (SARS) infection. In some embodiments, the viral lung infection is a Middle East respiratory syndrome (MERS) infection. In some embodiments, the viral lung infection is an influenza virus infection. In some embodiments, the viral lung infection is an Ebola virus infection. In some embodiments, the viral lung infection is a rabies infection. In some embodiments, the viral lung infection is a West Nile virus infection. In some embodiments, the viral lung infection is a dengue virus infection. In some embodiments, the viral lung infection is a respiratory syncytial virus (RSV) infection. In some embodiments, the viral lung infection is a Zika virus infection.

[0088] In some embodiments, the active agent is selected from the group consisting of (a) MSC-NTF, (b) EXO-MSC-NTF, and (c) a combination of MSC-NTF and EXO-MSC-NTF. In some embodiments, the active agent is MSC-NTF. In some embodiments, the active agent is EXO-MSC-NTF. In some embodiments, the active agent is a combination of MSC-NTF and EXO-MSC-NTF.

[0089] In some embodiments, the active agent is a combination of MSC and EXO-MSC. In some embodiments, the active agent is a combination of MSC-NTF and EXO-MSC-NTF.

[0090] In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient at least 4 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient at least 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 2 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 3 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 4 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient less than or equal to 2 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient less than or equal to 3 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient less than or equal to 4 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient less than or equal to 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 1 to 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 2 to 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 3 to 5 times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient 4 to 5 times.

[0091] In some embodiments, EXO-MSC-NTF, compared to its corresponding EXO-MSC, (i) substantially contains less of at least one protein selected from the group consisting of A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q92598, P05023, and P62873, or (ii) substantially contains more of at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

[0092] In some embodiments, EXO-MSC-NTF, compared to its corresponding EXO-MSC, (i) substantially contains less of at least one protein selected from the group consisting of A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q92598, P05023, and P62873, and (ii) substantially contains more of at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

[0093] In some embodiments, EXO-MSC-NTF contains (i) 2.46 - 2.73 pg of LIF protein per 1 μg of total protein mass, (ii) 5.33 - 7.48 pg of AREG protein per 1 μg of total protein mass, (iii) 0.45 - 0.78 pg of HGF protein per 1 μg of total protein mass, or (iv) 0.027 - 0.065 pg of TSG6 protein per 1 μg of total protein mass.

[0094] In some embodiments, EXO-MSC-NTF comprises (i) 2.46 - 2.73 pg of LIF protein per 1 μg of total protein mass, (ii) 5.33 - 7.48 pg of AREG protein per 1 μg of total protein mass, (iii) 0.45 - 0.78 pg of HGF protein per 1 μg of total protein mass, and (iv) 0.027 - 0.065 pg of TSG6 protein per 1 μg of total protein mass.

[0095] The terms "mesenchymal stem cell", "mesenchymal stromal cell", "multipotent stromal cell", "MSC", or "MSCs" are used interchangeably to refer to adult cells that have not yet terminally differentiated, adult cells that can divide to produce stem cells, or adult cells that can irreversibly differentiate to produce cells of the mesenchymal cell lineage or undergo transdifferentiation to differentiate into other non-mesodermal cell types, such as cells of the neuronal cell lineage.

[0096] The source of MSCs may be a healthy subject, the subject being treated, a donor immunologically matched to the subject being treated, or a donor not immunologically matched. In some embodiments, the source of MSCs can be a subject suffering from a neurodegenerative disease. In some embodiments, MSCs include autologous cells. In another embodiment, MSCs include allogeneic cells. As exemplified herein, EXO-MSC and EXO-MSC-NTF express little to no MHC-I and MHC-II molecules, so immunological matching between exosomes and human recipients may be redundant.

[0097] Mesenchymal stem cells (MSCs) are found in almost all tissues and can be isolated from a variety of tissues. Bone marrow (BM) is the most widely recognized source of MSCs, but recent studies have revealed other MSC sources such as adipose tissue (AT), placenta, dental pulp, synovium, peripheral blood, oral mucosa, periodontal ligament, endometrium, umbilical cord (UC), and umbilical cord blood (UCB). Indeed, evidence suggests that MSCs may be substantially present in vascular tissues throughout the body.

[0098] In some embodiments, the MSCs described herein are isolated from any tissue in which they are present. In some embodiments, tissues from which MSCs are isolated include, but are not limited to, bone marrow, adipose tissue, placenta, dental pulp, synovium, peripheral blood, oral mucosa, periodontal ligament, endometrium, umbilical cord Wharton's jelly, and umbilical cord blood.

[0099] In some embodiments, the MSCs are selected from the group consisting of bone marrow MSCs, adipose cell MSCs, dental pulp MSCs, placental MSCs, synovial MSCs, peripheral blood MSCs, oral mucosa MSCs, periodontal ligament MSCs, endometrial MSCs, umbilical cord Wharton's jelly MSCs, and umbilical cord blood MSCs.

[0100] The term "extracellular vesicles" (EVs) refers to a heterogeneous population of vesicles of cellular origin that are derived from the endosomal compartment (exosomes) or result from shedding from the cell membrane. Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released from essentially all prokaryotic and eukaryotic cells. The diameter of EVs ranges from those close to the size of the physically smallest unilamellar liposomes (about 20 - 30 nanometers) to those over 10 micrometers, although the majority of EVs are less than 200 nm. EVs are defined as exosomes, microvesicles, and apoptotic bodies based on size and synthetic pathway. These carry cargo such as proteins, nucleic acids, lipids, metabolites, and even cell organelles from the parent cell. Exosomes are small EVs (in the range of 30 - 150 nm) and are generated by the invagination of the endosomal membrane that forms intraluminal vesicles within multivesicular bodies (MVBs).

[0101] In some embodiments, the isolated exosome population further comprises one or more neurotrophic factors (NTFs) selected from the group consisting of hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-inducible gene 6 protein (TSG-6; also known as TNF-stimulated gene 6 protein), bone morphogenetic protein 2 (BMP2), fibroblast growth factor 2 (FGF2), and any combination thereof. In further related aspects, the isolated exosome population further comprises one or more miRNA molecules selected from the group consisting of miRNA (miR)-3663-3p, miR-132-3p, miR-150-3p, miR-762, miR-4327, miR-3665, miR-34a-5p, miR-1915, miR-34a-39, miR-34b-5p, miR-874, miR-4281, miR-1207-5p, miR-30b-5p, miR-29b-3p, miR-199b-5p, miR-30e-5p, miR-26a-5p, miR-4324, and any combination thereof, or the isolated exosome population lacks one or more miRNA molecules selected from the group consisting of miR-503, miR-3659, miR-3529-3p, miR-320b, miR-1275, miR-3132, miR-320a, miR-495, miR-181b-5p, miR-222-3p, miR-424-5p, miR-4284, miR-574-5p, miR-143-3p, miR-106a-5p, miR-455-3p, miR-20a-5p, miR-145-5p, miR-324-3p, miR-130b-3p, miR-1305, miR-140-3p, and any combination thereof, or a combination thereof.

[0102] As used herein, the term "about" means defining ±10% of the indicated numerical value. For example, the expression "about 10" means "9 to 11".

[0103] The above embodiments are only intended for illustration. Those skilled in the art can make various changes, modifications, and variations to the specific embodiments. The scope of the claims should not be limited by the specific embodiments described in this specification, but should be interpreted in a manner consistent with the specification as a whole.

[0104] Example

[0105] Example 1: Bone marrow collection for isolating mesenchymal stem cells (MSCs)

[0106] The purpose of this protocol is to describe the aspiration procedure of donor bone marrow (BM) for isolating mesenchymal stem cells to be used in the treatment of patients with viral lung infections that cause severe respiratory disorders, such as severe coronavirus disease 2019 (NCP) caused by COVID-19 or other viral lung infections.

[0107] Before the bone marrow aspiration (BMA) procedure, a document reporting the donor's test results for HIV1, HIV2, HBV, HCV, HTLV, syphilis, and COVID-19 was submitted. Positive test results that would exclude the bone marrow aspiration procedure include, but are not limited to, tests for anti-HIV-1, anti-HIV-2, hepatitis B virus (HBV; surface and core antigens), and hepatitis C virus (HCV) performed within one week of the bone marrow aspiration procedure.

[0108] Human bone marrow (80 - 120 ml) was collected by a physician using a 20-ml syringe pre-filled with approximately 1 ml of heparin-containing solution (heparin stock solution, USP, 350 units / ml in PlasmaLyte) through multiple punctures on both sides of the iliac crest of the pelvic bone according to the standard procedures of the medical center (under sedation, epidural anesthesia, or general anesthesia as necessary).

[0109] Example 2: Proliferation of MSCs

[0110] In the first stage of the manufacturing process, mononuclear cells (MNCs) are separated from the whole bone marrow by Ficoll density gradient centrifugation.

[0111] hMSCs were concentrated from mononuclear cells (MNCs) in vitro (within a two-chamber cell stack manufactured by Corning) by utilizing their ability to adhere to plastic. To prevent potential risks of infection and host immune response, the manufacturing process was carried out in a xenofree growth medium containing 10% human platelet lysate (PL) and a designated proliferation medium (PM). For the first 16 - 24 hours, the cells were seeded into the PM in a two-chamber cell stack (tissue culture vessel) at 37°C / 5% CO 2 and incubated. At this stage, the MSCs that adhered to the plastic attached to the surface of the cell stack, and the non-adherent mononuclear cells floated in the supernatant. The PM was replaced with fresh PM (passage number 0: P0). During passage number 0, the hMSC medium was exchanged 4 - 6 times. After a maximum of 15 days, the P0 MSCs were harvested and cryopreserved.

[0112] After harvesting the P0 MSCs and prior to cryopreservation, MSC cultures were sampled for in-process sterility testing, identified by flow cytometry, and tested for the presence of mycoplasma.

[0113] MSCs were identified by phenotypic analysis of cell surface markers by flow cytometry. hMSCs are characterized by the expression of CD73, CD90, and CD105 on the cell surface (>95% positive). To confirm the purity of the cell population and exclude the presence of hematopoietic cell contamination, these cells must lack the expression of CD14, CD34, CD45, and HLA-DR as measured by flow cytometry (<5%). The MSCs comply with the specifications.

[0114] 15×10 6 Eighteen cryotubes containing 15×10 cells / tube were cryopreserved in the gas phase of a liquid nitrogen freezer (-196°C) that provides stable cryogenic storage. The gas-phase liquid nitrogen freezer maintains low temperature even during filling and sample collection cycles.

[0115] After cryopreserving the MSCs of P0, the cells were thawed and seeded for proliferation (passage number 1: P1). The thawed hMSCs were seeded in the proliferation medium (PM) in a two-chamber cell stack at a concentration of 1,000 cells / cm 2 for 7 - 8 days. The proliferation medium (PM) was changed every 3 - 4 days. After a maximum of 7 - 8 days, the P1 cells were harvested and optionally cryopreserved (passage number 1).

[0116] After harvesting the passage 1 cells and before cryopreserving them, MSC cultures were sampled for in-process sterility testing and mycoplasma testing.

[0117] 25×10 6 Cryotubes containing 12 cells / tube were cryopreserved in the gas phase of a liquid nitrogen freezer (-196 °C) that provides stable cryogenic storage.

[0118] After cryopreserving the MSCs of P1, the cells were thawed and seeded for proliferation (passage number 2: P2). The thawed hMSCs were seeded in the proliferation medium (PM) in a two-chamber cell stack at a concentration of 1,000 cells / cm 2 for 7 - 8 days. The proliferation medium (PM) was changed every 3 - 4 days. After a maximum of 7 - 8 days, the P2 cells were harvested and cryopreserved (passage number 2).

[0119] After harvesting the passage 2 cells and before cryopreserving them, MSC cultures were sampled for in-process sterility testing and mycoplasma testing.

[0120] 30×10 6 Cryotubes containing 28 cells / tube were cryopreserved in the gas phase of a liquid nitrogen freezer (-196 °C) that provides stable cryogenic storage. To produce one dose (100×10 6 cells) for one patient, 5 cryotubes are required. To produce a low-dose one-dose (20×10 6 cells) for one patient, 1 cryotube is required. Alternatively, 130×10 6Cryotubes containing individual cells / tubes are cryopreserved in the gas phase of a liquid nitrogen freezer (-196 °C) that provides stable cryogenic storage.

[0121] For patient treatment with allogeneic MSCs, the cells were thawed and the final product was prepared. After thawing of the MSCs, the cells were pooled, washed, and counted. Subsequently, the MSCs were filled into syringes and labeled. Samples of the MSC suspension were taken for the final bulk safety tests of sterility, Gram staining, and endotoxin. Alternatively, at the patient's bedside, cells were thawed from cryotubes containing 130 × 10 6 individual cells / tube and immediately administered to the patient. Visual inspection of the final product syringes was performed to confirm compliance with the specifications (the syringe is intact, the cell suspension is turbid and yellowish, and there are substantially no visible particles in the cell suspension).

[0122] Example 3: Production, Purification, and Characterization of EXO-MSCs Containing EXO-MSC-NTF

[0123] For scale-up of exosome production and improvement of yield, thawed MSCs (P0 or P1; see Example 2) were resuspended in proliferation medium (PM) and seeded directly in a Quantum cell growth system bioreactor (Terumo BCT) or seeded in cell stacks for reseeding in a PBS bioreactor system (PBS Biotech) and grown for several days.

[0124] Quantum

[0125] The Quantum cell growth system is a functionally closed, automated hollow fiber type bioreactor system. The bioreactor itself is composed of approximately 11,500 hollow fibers, and the total surface area in the capillary (IC) is 2.1 m 2It is. The fluid circuit of the Quantum system is designed around two fluid loops. One is the in-capillary (IC) loop, and the other is the loop of the extra-capillary (EC) part of the hollow fiber.

[0126] The PBS bioreactor system (PBS Biotech) is a vertical wheel-type single-use bioreactor that can provide uniform, low-shear, and scalable mixing over a wide range of workloads.

[0127] Exosome bioreactor cell culture

[0128] The first stage of the manufacturing process involves the separation of mononuclear cells (MNCs) from whole bone marrow using a fully automated, closed, and compact solution, Sepax2 (Cytiva), for separating MNCs by Ficoll density gradient centrifugation. The Quantum cell expansion system is seeded with either MNCs after Sepax separation, thawed P0 MSCs, or P1 MSCs grown in a cell stack.

[0129] Before seeding the cells, the bioreactor was coated with 5 - 10 mg of fibronectin for at least 4 hours to overnight, and the "Coat Bioreactor Task" was used to promote cell adhesion. After coating the bioreactor for 4 hours to overnight, the excess fibronectin was washed out from the bioreactor set, and the cell culture medium was introduced into the bioreactor set using the IC / EC Washout Task to replace the PBS solution with the PM growth medium in DMEM without adding antibiotics / antifungal agents.

[0130] Human mesenchymal stem cells (hMSCs) were concentrated from mononuclear cells (MNCs) in vitro in a Quantum bioreactor by taking advantage of their ability to attach to the surface of the hollow fibers. To prevent potential risks of infection and host immune response, the manufacturing process was carried out in a xenofree growth medium containing 10% human platelet lysate (PL without antibiotics / antifungals) and a designated proliferation medium (PM). For the first 16 - 24 hours, at 37°C / 5% CO 2 The cells were seeded into the proliferation medium (PM) in the Quantum system. At this stage, the MSCs attached to the hollow fibers adhered to the surface of the bioreactor, and the non - attached mononuclear cells floated in the supernatant. The PM was replaced with fresh PM (passage number 0: P0). After up to 15 days, the P0 MSCs were harvested, a part of which was cryopreserved (passage number 0), and the remaining part was reseeded into a new Quantum cell growth system bioreactor.

[0131] Twenty million P0 MSCs were transferred into the cell inlet bag of the Quantum bioreactor, and the total volume of the bag was increased to 100 mL using the proliferation medium (PM). Then, the bag was aseptically connected to the cell inlet line of the Quantum system, and the cells were loaded onto the IC side pre - coated with fibronectin of the bioreactor using the "Load Cells with Circulation" task. Fresh PM was added to the IC side of the bioreactor, and the cells were grown for 6 - 7 days using the "Cell Supply" task, which adjusted the IC inflow rate according to the rates of glucose consumption and lactate production in the system sampled daily from the sample port. After 6 - 7 days, the MSCs (passage number 1: P1) were harvested.

[0132] After harvesting the P1 MSCs and before cryopreserving them, MSC cultures were sampled for in - process sterility testing, identified by flow cytometry, and tested for the presence of mycoplasma.

[0133] MSCs were identified by phenotypic analysis of cell surface markers by flow cytometry. hMSCs are characterized by the expression of CD73, CD90, and CD105 on the cell surface. To confirm the purity of the cell population and exclude the presence of hematopoietic cell contamination, the cells must lack expression of CD14, CD34, CD45, and HLA-DR as measured by flow cytometry.

[0134] 15×10 6 Cryotubes containing cells / ml were frozen and stored in the vapor phase of a liquid nitrogen freezer (-196 °C), which provides stable cryogenic storage. The vapor phase liquid nitrogen freezer maintains the low temperature even during filling and sampling cycles.

[0135] After cryopreservation of MSCs at P1, the cells were thawed and seeded for expansion (passage number 2: P2). 20 million thawed and washed MSCs (cells were frozen and stored in liquid N 2 The cells (which had been cryopreserved in 500 ml at passages 0–2) were transferred to a cell infusion bag and the total volume of the bag was brought up to 100 mL using growth medium (PM). The bag was then sterilely connected to the cell infusion line of the Quantum system and the cells were loaded into the fibronectin-precoated IC side of the bioreactor using the “cell loading by circulation” task. Fresh PM was added to the IC side of the bioreactor and the cells were allowed to grow for 6–7 days using the cell feeding task, which adjusted the IC inflow rate according to the rate of glucose consumption and lactate production in the system, which were sampled daily from the sample port. After 6–7 days, the medium was replaced with medium without platelet lysate. To produce the maximum number of exosomes from the same cells, taking into account the glucose consumption rate and lactate production rate, the cell medium containing exosomes was collected 2–5 times in total every 48 h.

[0136] The PBS3 MAG bioreactor equipped with a 3-liter single-use container (PBS Biotech) was loaded with 100 - 200 g of Synthemax II low-concentration microcarriers (Corning), or adhesion-enhanced microcarriers (Corning). Subsequently, this container was filled with 1.8 L of cell culture medium (DMEM high glucose supplemented with 1 - 2% human platelet lysate, glutamine, pyruvate, 200 μM ascorbic acid, and heparin) and equilibrated overnight.

[0137] 70 - 80×10 6 70 - 80×10⁶ MSCs were inoculated into a single-use bioreactor container and allowed to adhere to the microcarriers for 20 - 60 minutes during the attachment phase, followed by agitation with a wheel impeller at low speed for 4 - 6 hours. After the attachment phase, the agitation speed of the wheel impeller was increased, and an additional 1.2 L of cell culture medium was added to the container to bring the total volume to 3 L, and the final human platelet lysate concentration was set to 10%.

[0138] EXO-MSC was generated by culturing MSCs under continuous agitation for 5 - 7 days while maintaining the final concentration of PL at 10% and performing a 50 - 80% medium change daily starting from day 3. On the final day of culture, the medium was exchanged to platelet lysate-free medium to harvest exosomes. The cell culture medium containing exosomes was harvested at the end of the manufacturing process or collected a total of 2 - 5 times every 48 hours.

[0139] After collection, all collected media were pooled to isolate exosomes.

[0140] EXO-MSC-NTF was generated by culturing cells under continuous agitation for 5 - 7 days while maintaining the final concentration of PL at 10% and performing a 50 - 80% medium change daily starting from the 3rd day. On the final day of culture, in order to collect exosomes 72 hours later, the growth medium was replaced with S2M differentiation medium (Dulbecco's Modified Eagle Medium (high glucose) (Sigma-Aldrich) containing 1 mM dibutyryl cyclic AMP (cAMP), 20 ng / ml human basic fibroblast growth factor (hBFGF), 5 ng / ml human platelet-derived growth factor (PDGF-AA), and 50 ng / ml human heregulin β1, with 200 μM ascorbic acid added). The exosome isolation steps were as follows and were the same for EXO-MSC and EXO-MSC-NTF. EXO-MSC and EXO-MSC-NTF were isolated and purified using tangential flow filtration (TFF). To remove microcarriers, first, the exosomes containing the medium were passed through a 100 μm separation bag, and then the medium was filtered through a 0.8 - 1.2 μ filter to remove cell debris. The exosomes containing the filtrate were collected under sterile conditions and subjected to tangential flow filtration (TFF) (Repligen).

[0141] TFF: 100 kDa, 300 kDa, or 500 kDa, 500 - 1000 cm 2 or 2,500 - 5,000 cm 2A molecular weight cut-off (MWCO) membrane with a filtration area (from Repligen) was used. A sample containing exosomes was continuously pumped through the membrane system and recycled. Low-molecular-weight free proteins not contained within or bound to the membrane vesicles passed through the membrane pores and eluted as the filtrate product, which was ultimately discarded. Molecules too large to pass through the membrane pores, such as exosomes (or larger microvesicles), continued to circulate as the residue. To further remove samples of contaminants smaller than the KDa of the MWCO membrane, the sample was subjected to diafiltration with 5 - 10 volumes. In the final filtration cycle, the sample was reduced to a volume of approximately 100 ml. Finally, the sample was sterilized with a 0.2 μm filter.

[0142] Nanoparticle tracking analysis

[0143] The amount and size of the particles were measured using ZetaView nanoparticle tracking analysis (ParticleMetrix), a laser scattering video microscope that tracks the movement of individual nanoparticles under Brownian motion. For each sample, five exposures were recorded at 11 measurement positions. The particle size was calculated according to the Stokes-Einstein relation using ZetaView software (ZetaView 8.02.28).

[0144] FACS analysis

[0145] The phenotypic examination of exosomes was performed using the MACSPlex Exosome Kit, which enables the detection of 37 exosome surface epitopes and 2 isotype controls. This kit contains a mixture of various fluorescently labeled bead populations coated with specific antibodies that bind to each surface epitope. The 39 bead populations can be distinguished by differences in fluorescence intensity detected in the FITC and PE channels of a flow cytometer. Analysis of exosomes derived from BM-MSCs revealed high expression of tetraspanins (a conserved set of proteins expressed on exosomes, such as CD81, CD63, and CD9), MSC CD markers (CD44, CD29, CD49e), and negative expression of hematopoietic CD markers (CD4, CD19, etc.) and HLA-DR and HLA-ABC.

[0146] Example 4: Evaluation of the Efficacy of EXO-MSC Administration in an ARDS Mouse Model

[0147] The purpose of this study was to investigate the efficacy of bone marrow-derived mesenchymal stem cell exosomes (administered intratracheally or intravenously) in a mouse model of acute respiratory distress syndrome (ARDS), the main cause of death due to coronavirus.

[0148] By using animals in the ARDS model, the efficacy of EXO-MSC in suppressing clinical symptoms caused by the inflammatory response can be investigated, enabling the development of this treatment method for ARDS. The LPS-induced ARDS model is an accepted model for severe human acute respiratory diseases caused by coronavirus infection.

[0149] Administration was performed by intratracheal administration of EXO-MSC via an endotracheal tube (intratracheal administration) or intravenous administration of EXO-MSC at a concentration of 2.0×10 10 vesicles / 1 ml (Table 1).

[0150] Animals; Selection of the animal model: LPS-induced ARDS. Species / strain: BALB / c mice. Sex / Number / Age: Female, n = 60, 8 weeks old.

[0151]

Table 1

[0152] Induction of ARDS: BALB / c mice were anesthetized and orally intubated with a sterilized plastic catheter, and 800 μg of LPS dissolved in 50 μL of standard PBS was administered intratracheally. The same amount of PBS was injected into naive mice (without LPS administration, test group 6) as a control.

[0153] Administration: Daily administration of EXO-MSC via an intratracheal tube, or intravenous administration at a concentration of 2.0×10 10 particles / 1 ml. Administration was started 3 hours after LPS administration.

[0154] Sample collection: Blood sampling for whole blood hematological analysis for cell counting and serum analysis. For T lymphocytes, B lymphocytes, eosinophils, neutrophils, dendritic cells, monocytes / macrophages, measurement of total bronchoalveolar lavage (BAL) fluid and measurement of cell percentages by FACS were performed. Also, BAL fluid samples were analyzed for the presence of inflammatory cytokines. Lungs were excised from all animals sacrificed on day 3 for histopathology H&E.

[0155] Histological evaluation: Quantitative analysis of acute lung injury (ALI) was performed using a severity scoring scale of 0 - 2 based on the American Thoracic Society Acute Lung Injury in Animals Study Group (「Matute-Bello et al., Am J Respir Cell Mol Biol 44;725-738, 2011」; incorporated herein by reference).

[0156] 1. Neutrophils: Not visible in the field - Score 0; 1 - 5 neutrophils - Score 1; More than 5 neutrophils - Score 2. 2. Fibrin: Invisible in the field - Score 0; A single intact band of fibrin in the air cavity - Score 1; Multiple eosinophilic membranes - Score 2. 3. Thickening of the alveolar wall: Only septal thickening more than twice normal due to technical artifacts was considered. Less than twice - Score 0; Twice to four times - Score 1; More than four times - Score 2.

[0157] Figure 1 shows the histopathological results of the lungs of representative mice in Test Groups 1 - 6 (see Table 1).

[0158] Figure 2A shows the group scores of alveolar wall thickening based on the histopathological results when exosomes were administered intratracheally to Test Group 1 ("No EVs" = "LPS + PlasmaLyte"), Test Group 2 (EV (EVs) = EXO - MSC). Statistically significant differences were observed, demonstrating that exosome therapy is effective in reducing alveolar wall thickening. Figure 2B shows the total acute lung injury scores based on the histopathology when exosomes were administered intratracheally to Test Group 1 ("No EVs" = "LPS + PlasmaLyte"), Test Group 2 (EV (EVs) = EXO - MSC). Statistically significant differences were observed, demonstrating that exosome therapy is effective.

[0159] Figure 3 shows the serum concentrations of IL - 1β (Figure 3A), IL - 6 (Figure 3B), MCP - 1 (Figure 3C), IFN - γ (Figure 3D), TNF - α (Figure 3E) in mice administered EXO - MSC intratracheally and mice administered PlasmaLyte. Statistically significant differences were observed ( * p < 0.05), demonstrating that exosome therapy is effective in reducing the serum concentrations of cytokines.

[0160] Figure 4 shows the concentrations of IL-1β (Figure 4A), IL-6 (Figure 4B), IP-10 (Figure 4C), IFN-γ (Figure 4D), TNF-α (Figure 4E), MCP-1 (Figure 4F), and IL-1α (Figure 4G) in the lung fluid of mice administered with EXO-MSC and mice administered with plasma lite. Statistically significant differences were observed, demonstrating that exosome therapy is effective in reducing the concentration of cytokines in the lung fluid.

[0161] Figure 5 shows the blood neutrophil concentrations in healthy animals (control group), LPS control group, and LPS + EXO-MSC-IV administration group. A statistically significant increase in blood neutrophil concentration was observed in the LPS control, while the effect of LPS was attenuated in the EXO-MSC administration group.

[0162] Example 5: Mouse Model of LPS-Induced Acute Lung Injury

[0163] The purpose of this study was to investigate the effectiveness of bone marrow-derived mesenchymal stem cells or EXO-MSC-NTF (intratracheal administration) in a mouse model of acute respiratory distress syndrome (ARDS), which is the main cause of death due to coronavirus. The LPS-induced ARDS model is an accepted model for severe human acute respiratory diseases caused by coronavirus infection.

[0164] Administration was performed by daily intratracheal administration via a tube of MSC or EXO-MSC-NTF at a concentration of 2.0×10 10 particles / 1 ml (Table 2).

[0165] Induction of ARDS: BALB / c mice were anesthetized, and a sterilized plastic catheter was orally intubated. 800 μg of LPS dissolved in 50 μL of standard PBS was intratracheally administered to test groups 1-3. Naïve mice (no LPS administration, test group 4) were injected with the same amount of PBS as a control.

[0166] Administration: 2.0×10 10Vehicle / daily administration of EXO-MSC via an endotracheal tube at a concentration of 1 ml of EXO-MSC or EXO-MSC-NTF. Administration was initiated 3 hours after LPS administration.

[0167]

Table 2

[0168] Figure 6 shows the total severity scores of acute lung injury in test groups 1-4. As shown, administration of EXO-MSC-NTF significantly protected the mice from the effects of LPS.

[0169] Figure 7 shows the scores for fibrin (Figure 7A), alveolar wall thickness (Figure 7B), and neutrophils (Figure 7C) in test groups 1-4. As shown, administration of EXO-MSC-NTF significantly reduced the effects of LPS on fibrin and alveolar wall thickness.

[0170] Figure 7 further shows the number of neutrophils in lung sections of test groups 1-4 (Figure 7D). As shown, administration of EXO-MSC-NTF significantly decreased the number of infiltrating neutrophils in the lung after LPS administration. Furthermore, there was no significant difference in the number of neutrophils after EXO-MSC-NTF administration compared to the number of neutrophils in mice not administered LPS.

[0171] Figure 8 - Histopathology: Shows a multifocal distribution of mainly neutrophil (acute) perivascular infiltration. Fibrin deposition is mild, and the alveolar wall is thickened in the affected area. Test group 3 (Figure 8C) shows moderate to severe lung injury with an average respiration (Resp) of 4.4. Test groups 1 (Figure 8A) and 2 (Figure 8B) showed moderate lung injury of 3.6 and 2.5, respectively. Test group 4 (Figure 8D) showed a very low score with an average respiration (Resp) of 0.3.

[0172] Figure 9 shows the oxygen saturation in test groups 1-4. As shown, administration of EX0-MSC-NTF or EXO-MSC significantly reduced the effects of LPS.

[0173] Figure 10 shows the concentrations of IFN-γ (Figure 10A) and IL-6 (Figure 10B) in the BAL fluid of Test Groups 1-4. As shown, administration of EXO-MSC-NTF significantly reduced the effect of LPS on the concentrations of IFN-γ and IL-6.

[0174] Figure 11 shows the BAL fluid concentrations of IL-10 (Figure 11A) and RANTES (Figure 11B) in Test Groups 1-4. As shown, administration of EXO-MSC-NTF significantly reduced the effect of LPS on the concentrations of IL-10 and RANTES.

[0175] Figure 12 shows the BAL fluid concentration of TNF-α in Test Groups 1-4. As shown, administration of EXO-MSC-NTF significantly reduced the effect of LPS on the concentration of TNF-α.

[0176] The severe form of COVID-19 is associated with thrombotic coagulation disorders. Its etiology involves the effect of the virus on the immune system and the downregulation of ACE2, which causes an increase in angiotensin II concentration. The increase in inflammatory cytokines and angiotensin II are both known as factors that induce tissue factor (TF) and activated neutrophils. Tissue factor (TF) may be an important mediator associated with the occurrence of thrombus phenomena in COVID-19.

[0177] Another coagulation factor, thrombin-antithrombin complex (TAT), has been found to be higher in non-survivors than in survivors in the early and middle stages of the disease, which reflects the excessive production of thrombin. The concentrations of tissue factor (TF) and TAT were examined using ELISA analysis in the sera and BALF of ARDS mice administered EXO-MSC or EXO-MSC-NTF.

[0178] As described in Example 3 above, MSCs are induced to differentiate into MSC-NTFs (MSCs that secrete neurotrophic factors) using a medium-based approach. To that end, MSCs are cultured in a medium containing (i) 1 mM dibutyryl cyclic AMP (cAMP), (ii) 20 ng / ml human basic fibroblast growth factor (hbFGF), (iii) 5 ng / ml human platelet-derived growth factor (PDGF-AA), and (iv) 50 ng / ml human heregulin β1.

[0179] The body weight of the animals was measured daily, and animals were excluded from the study if their body weight decreased by 20% from the baseline or if their body weight decreased by more than 10% during the measurement period. In addition, animals were excluded from the study if any of severe dehydration, lack of exercise, skin lesions, persistent tremors, or respiratory failure was observed. During the study period, the animals had free access to food and drinking water.

[0180] To measure the content of specific proteins in MSC-derived small extracellular vesicles (sEV, EXO-MSC), 1 ml of the sEV concentrated fraction was precipitated using ExoQuick-CG (SBI, USA). The EV pellet was lysed using M-PER mammalian protein extraction reagent (ThermoFischer, USA), and a 1:200 protease inhibitor cocktail set III, EDTA-free (Calbiochem) was added. After incubation at room temperature for 10 minutes, the lysate was frozen and then thawed twice to completely dissolve it. The protein concentration of the lysate was measured using a BCA kit (ThermoFischer, USA) and used for ELISA analysis at a concentration of 60 - 75 μg / ml. The concentrations of AREG and LIF were measured using a Quantikine kit (R&D Systems, Minneapolis, MN, USA, catalog number: DAR001, DLF00B). The concentrations of HGF and TSG-6 were measured using ELISA kits (catalog numbers: ELH-HGF-CL-1, ELH-TSG6-1) manufactured by RayBiotech, USA. The signals were quantified using a Sunrise plate reader and Magellan Software V7.2 (Tecan, Switzerland).

[0181] The immunomodulatory properties of EXO-MSC and EXO-MSC-NTF were evaluated in vitro by examining the suppression of cytokine secretion by peripheral blood mononuclear cells (PBMC) in response to activation by phytohemagglutinin (PHA). PBMC (5×10 5 cells) were stimulated with 10 μg / mL of PHA and cultured in medium with EXO-MSC or EXO-MSC-NTF (2×10 9 particles) for 4 days. IFN-γ and TNF-α in the culture supernatant were measured using a commercially available ELISA (DuoSet ELISA manufactured by R&D Systems, Minneapolis, MN, USA) read at 450 nm using a Sunrise plate reader and analyzed with Magellan Software V7.2 (Tecan, Switzerland).

[0182] When sEV, EXO-MSC, or EXO-MSC-NTF was added to activated PBMCs, the secretion of IFN-γ (Figure 13A) and TNF-α (Figure 13B) was suppressed. There was no significant difference in the ability of EXO-MSC and EXO-MSC-NTF to suppress IFN-γ secretion, but EXO-MSC-NTF significantly suppressed the secretion of TNF-α.

[0183] To investigate the differences between EXO-MSC and EXO-MSC-NTF that may contribute to the superior effects of EXO-MSC-NTF administration, the differences in the protein cargo of EXO-MSC and EXO-MSC-NTF from three independent donors were evaluated. By ELISA measurement, it was revealed that EXO-MSC-NTF had more than 16-fold more AREG and more than 3-fold more LIF compared to EXO-MSC (Figure 14A, Figure 14B; p = 0.013 and p = 0.015, respectively). In addition, HGF and TSG-6 were found to be present in both types of EVs, but there was no significant difference (Figure 14C, Figure 14D).

[0184] Table 3 summarizes the differences in the main protein cargo of EXO-MSC and EXO-MSC-NTF.

[0185]

Table 3

[0186] Example 6: Administration of mesenchymal stem cells (MSC) and mesenchymal stem cell exosomes (EXO-MSC) for severe coronavirus disease 2019 (COVID-19) pneumonia (NCP).

[0187] Primary objective: To evaluate the safety, tolerability, and efficacy of intravenous administration of MSC and / or EXO-MSC in severe NCP. MSC-NTF and EXO-MSC-NTF may be used instead of MSC and EXO-MSC.

[0188] Secondary objectives: Evaluate the efficacy of MSCs and EXO-MSCs using the improvement in the Critical Treatment Index (CTI). Evaluate changes in BAL and blood biomarkers after MSC administration. Evaluate the efficacy of intravenous administration of MSCs for severe NCP due to COVID-19 using (a) the duration of ventilator-free days during the study period or (b) overall survival / mortality. Evaluate changes in cellular and soluble biomarkers after MSC administration.

[0189] This study is a randomized, parallel-group, non-blinded trial conducted at multiple study sites in up to 60 subjects who developed severe novel coronavirus pneumonia (NCP) due to COVID-19 at the time of screening visit. After obtaining informed consent and signature on the informed consent document, all subjects were randomly assigned to this study and observed for a total of 28 days (one month).

[0190] Subjects eligible and meeting the exclusion criteria will be randomly assigned to one of three cohorts: intravenous administration of MSCs (80 - 100 M MSCs / 4 ml), intravenous administration of EXO-MSCs (at least 1.0×10 10 individual EXO-MSCs / 10 ml), or intravenous administration of a combination of MSCs and EXO-MSCs, and administered on days 1, 2, 3, 4, and 5, or days 1, 3, and 5.

[0191] After 3 or 5 days of administration, subjects will be followed up for up to 28 days. Safety parameters, physiological parameters, and biomarkers of the study will be obtained.

[0192] This study consists of a 5-day administration period followed by a follow-up period until day 28 (about one month, Figure 15). Administration will be performed in the hospital's acute care unit or intensive care unit (ICU). Subjects will be evaluated daily after each administration. After 3 or 5 days of administration, all subjects will be followed up for up to 28 days for evaluation of primary efficacy and safety. Therefore, each subject will be followed up for a total of about 28 days (one month) from the first visit.

[0193] Eligible subjects who meet the eligibility criteria / exclusion criteria are randomly assigned to one of the six cohorts in Table 4 and receive the administration.

[0194]

Table 4

[0195] Procedure for intravenous administration of MSC: Procedure for intravenous administration of MSC (80 - 100M MSC / 4ml). Use a product filled with 4ml of cell suspension in a 5ml syringe. Inject the cell suspension from the syringe into a 100ml PlasmaLyte A bag and infuse it into the subject over 1 hour.

[0196] EXO - MSC intravenous administration procedure: EXO - MSC (at least 1.0×10 9 exosomes / 10ml) intravenous administration procedure. Use a product filled with 10ml of exosomes in a 10ml syringe. Inject the exosomes from the syringe into a 100ml PlasmaLyte A bag and infuse it into the subject over 1 hour.

[0197] Procedure for intravenous administration of the combination of MSC and EXO - MSC: Procedure for intravenous administration of the combination of MSC (80 - 100M MSC / 4ml) and EXO - MSC (at least 1.0×10 9 exosomes / 10ml). Inject the MSC product and the exosome product into separate 100ml PlasmaLyte - A bags, and infuse them into the subject over 1 hour with an interval of at least 2 hours.

[0198] It should be understood that cells (MSC, MSC - NTF) can be administered by intravenous administration, while exosomes (EXO - MSC, EXO - MSC - NTF) can be administered by intravenous administration, intratracheal administration, and nasal administration (nasal inhalation).

[0199] Screen the subjects and enroll eligible subjects. Follow up each subject for approximately 28 days and conduct an evaluation of efficacy and safety over approximately 28 days or at the end of the trial.

[0200] Intravenous administration of MSC (80 - 100M MSC / 100 ml) and combination with intravenous administration of EXO - MSC (at least 1.0×10 9 exosomes / 10 ml), or combination of intravenous administration of MSC (80 - 100M MSC / 100 ml) and EXO - MSC (at least 1.0×10 9 exosomes / 10 ml) are administered repeatedly (for 5 consecutive days or for 3 days every other day).

[0201] This dosage has been shown to be safe for intrathecal transplantation of 100 - 125×10 6 MSC - NTFs in more than 200 ALS patients and MS patients. EXO - MSC is derived from the same MSC cell source.

[0202] This trial was conducted on patients hospitalized with severe novel coronavirus pneumonia (NCP) caused by COVID - 19. To participate in this trial, all subjects are required to meet all eligibility criteria and not fall under the exclusion criteria.

[0203] Subjects who met all of the following criteria were permitted to participate in the trial. 1. Men and women aged 18 years or older and less than 75 years old at the screening examination. 2. Those in whom 2019 - nCoV infection was confirmed by clinical examination by reverse transcription polymerase chain reaction (RT - PCR) from any diagnostic sampling source. 3. Those in whom pneumonia consistent with COVID - 19 was confirmed by baseline chest computed tomography. 4. (1) Dyspnea (RR of 30 breaths / min or more), (2) Finger oxygen saturation at rest of 93% or less, (3) Arterial oxygen partial pressure (PaO 2 ) / Oxygen absorption concentration (FiO 2) is 300 mmHg or less, and (4) in the lung imaging examination, the focus progression exceeds 50% within 24 to 48 hours, and those who meet any one of them. 5. Those with ARDS associated with COVID-19 infection. 6. Those who have a medical need for endotracheal intubation and mechanical ventilation. 7. Those who the doctor determines are receiving maximum intensive medical treatment.

[0204] Or, the main eligibility criteria are as follows: 1 to 5 below. 1. Men and women aged 18 or older and under 75 years old. 2. Those in whom 2019-nCoV infection has been confirmed by clinical examination by reverse transcription polymerase chain reaction (RT-PCR) from any diagnostic sampling source. 3. Those with an acute onset of ARDS including the following (1) to (5) defined by the Berlin criteria. (1) Those in whom pneumonia or deterioration of respiratory symptoms are recognized within one week from a known clinical disorder. (2) Those in whom bilateral lung shadows not explained by pleural effusion, lobar / lung collapse, or nodules are recognized on chest X-ray or CT scan. (3) Those in whom pulmonary edema not sufficiently explained by heart failure or fluid overload is recognized. (4) PaO 2 / FiO 2 ratio is less than 300 mmHg, and those with hypoxemia defined as such are recognized. 4. Those in whom radiological lung changes (consolidation, ground-glass shadows, or bilateral lung infiltrates) consistent with COVID-19 ARDS are recognized by baseline high-resolution chest computed tomography (HRCT) obtained within 5 days from the start of administration. 5. Those in whom respiratory failure defined as blood oxygen saturation (SpO 2 ) less than 93% is recognized.

[0205] Subjects who meet any of the following criteria during the screening evaluation were excluded from participation in the study. 1. If they have previously received stem cell therapy. 2. If there is a history of malignant tumor within the past 5 years. However, it excludes localized cutaneous cancer of non-melanoma where there is no metastasis, significant invasion, or recurrence within 3 years from the (first visit screening test). 3. If currently using immunosuppressive agents or have used such drugs within 6 months after trial registration. However, this does not include the use of steroid drugs for therapeutic purposes or other treatments recognized as necessary for the management of COVID-19. 4. If the patient is a pregnant or lactating woman. 5. If informed consent cannot be obtained from the patient or the authorized family member.

[0206] Or, the main exclusion criteria are as follows: 1 - 5 below. 1. If informed consent cannot be obtained from the patient or the authorized family member. 2. If currently using chronic immunosuppressive agents or have used such drugs within 6 months after trial registration. However, this does not include the use of steroid drugs for therapeutic purposes or other treatments recognized as necessary for the management of COVID-19. 3. If the patient is a pregnant or lactating woman. 4. If the patient has previously received stem cell therapy. 5. If the patient is an organ transplant recipient.

[0207] Test evaluation

[0208] Bronchoalveolar lavage and blood sampling for biomarker evaluation: Bronchoalveolar lavage and serum sample collection for biomarker detection.

[0209] COVID virus load test: Nasopharyngeal swab (following the hospital protocol) to confirm the genome of the COVID virus.

[0210] Chest high-resolution CT scan: High-resolution computed tomography (HRCT) of the chest following the hospital protocol.

[0211] Clinical examination safety test: The clinical examination safety test monitors patients during their 1st to 9th visits to the hospital through the test.

[0212] Hematology: Complete blood count (CBC) (red blood cells [RBC] (index), white blood cells [WBC] (fraction and platelet count), hemoglobin [Hb], hematocrit [Ht]).

[0213] Serum pregnancy test drug: hCG

[0214] Blood biochemistry: Sodium (Na), potassium (K), calcium (Ca), bicarbonate (HCO 3 )), blood urea nitrogen (BUN), creatinine (Cr), glucose (Gluc), chloride (Cl), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), total bilirubin, aspartate aminotransferase (glutamic oxaloacetic transaminase) (AST[GOT]), alanine aminotransferase (glutamic pyruvic transaminase) (ALT[GPT]), alkaline phosphatase (ALP), uric acid.

[0215] Coagulation: Prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR).

[0216] Urine test: Specific gravity, pH, glucose, protein, ketone bodies, blood.

[0217] The measured values of vital signs (such as blood pressure, body temperature, pulse, respiratory rate, etc. after sitting for at least 3 minutes) are monitored at the time of screening (1st visit to the hospital) and at all visits until the last visit to the hospital.

[0218] At the 1st visit to the hospital, a standard 12-lead electrocardiogram is performed. The results of the electrocardiogram need to be read manually, preferably by a cardiologist, and the results should be entered into the electronic case report form (eCRF).

[0219] Daily assessment: Safety and adverse events, vital signs, laboratory evaluations (CBC and differential, platelet count, BUN, creatinine, LDH, PT, PTT, INR, ferritin, D-dimer, ALT, AST, pH, lactate, CK) including the clinical course of ARDS.

[0220] Respiratory physiological parameters (PaO 2 / FiO 2 ratio).

[0221] Blood / serum collection for biomarkers (before dosing on days 1, 2, 3, 4, 5 and approximately 6 hours after each dose).

[0222] Inflammatory markers: C-reactive protein (CRP), procalcitonin (PCT), and white blood cell differential.

[0223] Cytokines: IL-2, IL-6, IL-7, G-CSF, IP10, MCP-1, MIP-1a, IL-8 and TNF-α, IL-1-a and IL-1-b, IFN-y.

[0224] Biomarkers reflecting the paracrine activity of administered MSCs (VEGF, ANG-1, and KGF). Sequential Organ Failure Assessment (SOFA) score. Acute Physiology and Chronic Health Evaluation II (ApacheII) score.

[0225] On day 10 (in addition to daily assessments): Nasopharyngeal swab (NP) to confirm the presence or absence of the COVID virus genome. High-resolution chest computed tomography (HRCT) to evaluate changes in lung image abnormalities compared to baseline (before dosing, day 1). Serum / blood biomarker collection.

[0226] Day 28 (in addition to daily evaluations): Safety and adverse events (proportion of subjects with treatment-related adverse events evaluated according to CTCAE v4.0). Number of days without mechanical ventilation during the study period. Number of days without ICU stay during the study period. Overall survival / mortality (proportion of deaths due to all causes). Clinical Critical Treatment Index.

[0227] Hospital visit before administration

[0228] First hospital visit: Hospital visit for screening and randomization (Day 0).

[0229] Prior to conducting the study-specific screening assessment, it is necessary to obtain written informed consent (ICF) from the subject or the legal authorized representative (LAR).

[0230] Perform the following evaluations and procedures: Obtain the signed informed consent (acquired by the principal investigator (PI) and the sub-investigator (Sub-I)). Collect demographic data. Medical history. COVID medical history and diagnosis date. Nasopharyngeal swab (NP) to confirm the COVID virus genome. High-resolution chest computed tomography (HRCT). Bronchoalveolar lavage (BAL) for biomarker collection. Standard 12-lead electrocardiogram (ECG). Confirm past drug therapies. Measure vital signs (blood pressure, body temperature, pulse, respiratory rate, etc.). Respiratory variables (minute ventilation, respiratory rate, oxygenation index, PEEP value).

[0231] Clinical Critical Treatment Index: No activity limitation, discharged = Score 1; Activity limitation = Score 2; Hospitalized, no oxygen therapy = Score 3; Oxygen therapy with mask or nasal cannula = Score 4; Non-invasive ventilation or high-flow oxygen therapy = Score 5; Intubation and mechanical ventilation = Score 6; Mechanical ventilation + additional organ support - ECMO, CRRT, vasopressors = Score 7; Death = Score 8.

[0232] Blood sampling for hematological tests including hematological examination (CBC (hematology panel): hemoglobin, hematocrit, white blood cell count (and white blood cell differential), platelet count), coagulation tests (PT, PTT, INR), and biochemical evaluation (sodium, potassium, chloride, glucose, BUN, creatinine, bicarbonate, calcium, total bilirubin, AST, ALT, ALP, uric acid, total cholesterol, HDL, LDL).

[0233] Blood sampling (baseline) for serum biomarker analysis.

[0234] Blood sampling for serum pregnancy test (female subjects with possible pregnancy).

[0235] Urine test (specific gravity, pH, glucose, protein, ketone bodies, blood).

[0236] Determine the eligibility of the test and review the eligibility criteria / exclusion criteria.

[0237] Randomization of eligible subjects.

[0238] Visit for administration

[0239] Visit 1, 2, 3, 4, 5 (day 1, 2, 3, 4, 5), or visit 1, 3, 5 (day 1, 3, 5): For a subject to participate in the trial, they must continue to meet all eligibility criteria and not meet any exclusion criteria from the start date of the trial. If the clinical condition of the subject changes between screening (day 0) and the first visit (day 1), it is necessary to repeat some or all of the screening evaluations to assess the eligibility of the subject.

[0240] The pre - administration evaluation (up to 2 hours before administration) includes the following items: measurement of vital signs (systolic blood pressure (mmHg); body temperature, pulse, respiratory rate (per minute); SpO 2 Scale 1 (%), SpO 2Scale 2 (%) (such as use of air or oxygen). Clinical and radiological progression of ARDS. Safety and adverse events including consideration of concomitant medications. Blood samples are taken for blood tests (PT, PTT, INR), blood coagulation tests, and biochemical tests (sodium, potassium, chloride, glucose, BUN, creatinine). Collection of blood / serum for biomarker collection (about 6 hours after administration on days 1, 2, 3, 4, 5 or days 1, 3, 5). Inflammatory markers: C-reactive protein (CRP) and procalcitonin (PCT). Serum markers: IL-2, IL-6, IL-7, IL-8, G-SCF, IP-10, MCP-1, MIP-1A, TNF-α, IFN-γ, and IL-1-α. Bronchoalveolar lavage - total protein, albumin, IL-1β, IL-6, IL-8, TNF-α, SRAGE Immune cells: lymphocytes, neutrophils. Biomarkers reflecting the paracrine activity of administered MSCs (ANG-1, TSG-6, and KGF). Immune cells secreting cytokines: CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, CXCR3+ NK cells.

[0241] After administration, the following were performed on the subjects: Vital signs were monitored at 2 hours (±15 minutes), 8 hours (±15 minutes), and 20 hours (±30 minutes) after transplantation. Blood samples were taken at 20 hours (±30 minutes) after transplantation to evaluate biomarkers. Sequential Organ Failure Assessment (SOFA) score. Acute Physiology and Chronic Health Evaluation II (ApacheII) score. Glasgow Coma Scale (GCS) score. Consideration of adverse events (AE).

[0242] Follow-up after administration

[0243] 7th visit to the hospital (10th day) and 8th visit to the hospital (22nd day): At the follow-up visits on the 10th and 22nd days, the following were performed on the subjects. Review of concomitant medications. Review of adverse events. Measurement of vital signs (blood pressure, body temperature, pulse, respiratory rate, etc.). Confirmation of the COVID virus genome by nasopharyngeal swab (NP). High-resolution chest CT: Changes from baseline using standardized scoring. Bronchoalveolar lavage (BAL): Biomarker analysis compared to baseline. Blood / serum collection for serum biomarker analysis compared to baseline. Standard 12-lead electrocardiogram. Review of concomitant medications. Review of adverse events (AE).

[0244] Blood sampling for hematological tests (CBC (hematology panel): hemoglobin, hematocrit, white blood cell count (and white blood cell fractions), platelet count), coagulation tests (PT, PTT, INR), biochemical evaluation (sodium, potassium, chloride, glucose, BUN, creatinine, bicarbonate, calcium, total bilirubin, AST, ALT, ALP, uric acid, total cholesterol, HDL, LDL).

[0245] Urine test (specific gravity, pH, glucose, protein, ketone bodies, blood).

[0246] 9th visit to the hospital: Follow-up on the 28th day (±5 days). In the follow-up after the administration on the 9th visit to the hospital, the following were performed on all subjects: Review of concomitant medications. Examination of adverse events. Measurement of vital signs (blood pressure, body temperature, pulse, respiratory rate, etc.).

[0247] Blood tests for hematological examinations (CBC (hematology panel): hemoglobin, hematocrit, white blood cell count (and white blood cell differential), platelet count), coagulation tests (PT, PTT, INR), biochemical evaluation (sodium, potassium, chloride, glucose, BUN, creatinine, bicarbonate, calcium, total bilirubin, AST, ALT, alkaline phosphatase, uric acid, total cholesterol, HDL, LDL). Blood sampling for serological pregnancy tests (female subjects). Urine tests (specific gravity, pH, glucose, protein, ketones, blood). Safety and adverse events (proportion of subjects with administration-related adverse events evaluated according to CTCAE v4.0). Respiratory variables (minute ventilation, respiratory rate, oxygenation index, PEEP value). Sequential Organ Failure Assessment (SOFA) score. Acute Physiology and Chronic Health Evaluation II (Apache II) score. Glasgow Coma Scale (GCS) score.

[0248] Recorded the following: number of days of mechanical ventilation and number of subjects successfully weaned from mechanical ventilation. Length of stay in the ICU. Mortality rate, proportion of deaths from all causes. Number of days without organ failure (cardiovascular, coagulation, hepatic, renal) until day 28. SpO 2 / FiO 2 increased by 50 or more compared to its nadir. Time until oxygenation improved for at least 48 hours in the hospital. Clinical severity assessment index (improvement time).

[0249] No activity restriction, discharged = score 1; Activity restriction = score 2; In-hospital, no oxygen therapy = score 3; Oxygen therapy with mask or nasal cannula = score 4; Non-invasive ventilation or high-flow oxygen therapy = score 5; Intubation and mechanical ventilation = score 6; Mechanical ventilation + additional organ support - ECMO, CRRT, vasopressors = score 7; Death = score 8.

[0250] Safety Follow-up: For all subjects who have received administration or partial administration, safety and efficacy follow-up will be conducted for approximately 28 days. Follow-up observations will be made for adverse events (AE) and serious adverse events (SAE).

[0251] Information on the investigational drug. Overview of the mesenchymal stem cell (MSC) product

[0252] The MSC is provided in a ready-to-use dosing package with appropriate primary and secondary labels. The dosing package consists of one 5 mL syringe for intravenous administration. Each dosing package is 6 composed of a syringe containing 100×10

[0253] The syringe is capped with a stopper (not a needle). The 5 mL syringe for intravenous administration is packaged in a pouch.

[0254] The dosing package is delivered to the medical center by a transport system container designed to maintain a temperature of 2 - 8°C at the time of shipment. This product (investigational drug) is administered to patients within the set expiration date.

[0255] Alternatively, the dosing package consists of one cryotube containing 130×10 6 allogeneic MSC cells / tube for intravenous administration. The cryotube is shipped in the liquid nitrogen vapor phase and thawed at the patient's bedside.

[0256] The MSC is administered intravenously by injecting 4 mL of the cell suspension from the syringe into a bag filled with 100 mL of PlasmaLyte A and infused by drip over 1 hour.

[0257] The patient received a total of three infusions without any particular problems. As a result of clinical tests, a decrease in CRP and D-dimer was confirmed. The patient still required oxygen inhalation, but after administration, the oxygen saturation decreased from 40 L to 30 L, and the flow rate increased from 92% to 97%. The chest infiltration persisted. The PCR tests for the COVID-19 virus were both negative.

[0258] Overview of Mesenchymal Stem Cell Exosome (EXO-MSC) Product

[0259] EXO-MSC is provided in a ready-to-use administration package with appropriate primary and secondary labels. The administration package consists of one 10 mL syringe for intravenous administration. Each administration package is composed of a syringe that can be immediately injected and contains at least 1.0×10 9 EXO-MSC in 10 ml.

[0260] The syringe is capped with a stopper (not a needle). The 10 mL syringe for intravenous administration is packed in a pouch.

[0261] The administration package is delivered to the medical center by a transport system container designed to maintain a temperature of 2 - 8°C at the time of shipment. This product (investigational drug) is administered to the patient within the set expiration date.

[0262] Alternatively, the administration package consists of one cryotube containing MSC-exosomes in 10 ml. The cryotube is shipped with dry ice.

[0263] EXO-MSC-exosomes are administered intravenously by injecting 10 ml of cell suspension from the syringe into a bag filled with 100 ml of PlasmaLyte A and are infused by drip over 1 hour.

[0264] Previous Treatments and Concurrent Treatments

[0265] Prior treatment: Subjects who have received any cell therapy in the past will be excluded from this trial. To minimize the amount and impact of missing data, the principal investigator shall make every reasonable effort to collect the primary efficacy and safety data regarding subjects who had the administration discontinued or the trial discontinued. All medications taken before the first dose shall be recorded as pre-treatment medications.

[0266] Concomitant therapy and excluded therapy: Concomitant medications are those administered to the subject during or after the first dose. Record all concomitant medications. Currently using immunosuppressants or having used such medications within 6 months after trial registration will result in exclusion from the trial. However, the use of therapeutic agents such as corticosteroids deemed necessary for the management of COVID-19 is not included in this.

[0267] Safety reporting: In this trial, adverse events (AE) and serious adverse events (SAE) will be collected from the time of informed consent until the end of the trial (the 9th visit to the hospital or early termination visit). Results. In this trial, the following terms will be used. Fatal; un-recovered / unresolved; recovering / resolving; recovered / resolved; recovered / resolved with sequelae; unknown; clinically significant laboratory abnormality.

[0268] Laboratory abnormalities judged by the principal investigator of the clinical trial to be clinically significant shall be reported in the AE eCRF. Clinically significant abnormalities are those that have changed significantly since the screening visit to the hospital and have been confirmed by the judgment of the principal investigator of the clinical trial to require a change in management. This change includes further monitoring of clinical tests, initiation of other diagnostic tests or treatments, change in ongoing administration, or implementation of a new administration. As much as possible, record the etiology of the abnormal findings (e.g., anemia) in the eCRF. Repeat additional tests and / or other evaluations necessary to determine the importance and etiology of the abnormal results as clinically required.

[0269] Trial termination: Termination of the trial or the trial facility

[0270] During a clinical trial, situations may arise that prompt the termination of the trial or the withdrawal of a trial site from participation. Such situations include, but are not limited to, the following: Unanticipated, serious, or unacceptable risks are discovered in the subjects enrolled in the trial. The Data Safety Monitoring Board (DSMB) decides to recommend the interruption or termination of the trial. The sponsor decides to interrupt, terminate, or shorten the trial.

[0271] Situations where the conduct of the trial at a trial site is terminated include the following: The principal investigator of the trial fails to enroll eligible subjects in the trial. The principal investigator of the trial fails to comply with the guidelines of the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH)-Good Clinical Practice (GCP), or the guidelines and regulations of the Food and Drug Administration (FDA). There is a submission of false information from the trial site to the sponsor, clinical monitor, FDA, or Institutional Review Board (IRB). There is insufficient compliance with the requirements of the protocol. The interests of the principal investigator, their affiliated institution, or the personnel of the medical institution conducting the trial are in conflict and have an adverse impact on the integrity of the trial. The institution or IRB is subject to an investigation by the regulatory authority for legitimate reasons.

[0272] Withdrawal of Subjects from the Trial: Subjects may withdraw from the trial at any time during the trial period for any reason, without suffering any disadvantage. The principal investigator of the trial shall record the reason / situation for the discontinuation in the appropriate electronic case report form (eCRF) in a timely manner (preferably within 24 to 48 hours).

[0273] Subjects may discontinue the trial for any of the following reasons: Any reason related to safety or tolerability. If the subject so desires. If the principal investigator determines that it is appropriate for any reason. If the sponsor determines that it is appropriate for any reason.

[0274] Subjects who discontinue the trial for any reason shall undergo follow-up for all relevant evaluations regarding safety and efficacy, including the collection of clinical evaluations and clinical test results specified in this protocol.

[0275] The clinical trial facility must record the reason on the eCRF's trial end page in the electronic database. The record must include the date when the subject withdrew informed consent for the trial (the date when the trial was terminated), and the reason for the termination. The recorded date is considered the date of the last contact and is thus considered the last day of the trial for the subject. If the clinical trial facility becomes aware of an adverse event or serious adverse event (SAE) that occurred within 12 weeks of the last dose, despite the trial being terminated, the facility must record this in the adverse event log in the database.

[0276] Temporary suspension of the clinical trial: In the event of a serious adverse event (SAE), a major concurrent disease, or problems with cell manufacturing or patient visit schedules, the clinical trial may be temporarily suspended.

[0277] Clinical evaluation items

[0278] Primary evaluation item: Safety; The primary evaluation item is to evaluate the safety and tolerability when autologous MSCs and / or EXO-MSCs are administered intravenously for 5 consecutive days or for 3 consecutive days on alternate days. Safety and adverse events (the proportion of subjects with trial-related adverse events evaluated according to CTCAE v4.0).

[0279] Secondary evaluation items: Changes in BAL and blood biomarkers: The efficacy of MSCs and EXO-MSCs is evaluated by changes in BAL and blood biomarkers after administration. BAL samples and blood samples are collected according to the evaluation schedule, and biomarkers are evaluated before each administration throughout the trial to evaluate the relevance to the administration of MSCs and EXO-MSCs. Time to improvement of the clinical severity assessment index. Time until oxygenation improves for at least 48 hours in the hospital. Length of stay in the ICU. Mortality rate, proportion of deaths from all causes. Number of days on mechanical ventilation and number of subjects who successfully weaned from mechanical ventilation.

[0280] Statistical methods and determination of sample size

[0281] Determination of Sample Size: Formal calculation of the sample size is not performed. Data on the efficacy and safety of 60 subjects will provide information useful for the planning of future randomized clinical trials.

[0282] Statistical Methods: Summaries of continuous variables include sample size, mean, standard deviation, median, minimum, and maximum values. The minimum and maximum values are reported with the same precision as the raw values; the mean, standard deviation, and median are presented with one additional decimal place compared to those reported with the raw values. Summaries of discrete variables include frequency and percentage. All percentages are rounded to the second decimal place (i.e., XX.X%). Baseline visit is defined as the last non-missing measurement before the start of the investigational drug administration (the first administration at the first visit, day 0).

[0283] A detailed Statistical Analysis Plan (SAP) is completed before the first subject receives the administration.

[0284] Analysis Population

[0285] Primary, secondary, and exploratory efficacy endpoints are analyzed using the mITT (modified intent to treat) population and the Efficacy Evaluation Set (EE) population. In this trial, all subjects who received at least one administration and had at least one post-evaluation baseline are defined as the mITT population. Baseline is defined as the most recent evaluation before receiving the first administration at the second visit (day 1). The EE population is defined as a subset of the mITT population that received all 5 administrations and did not deviate from the important protocol that affects the efficacy evaluation. If the EE population is the same as or very similar to the mITT population, the analysis is performed only on the mITT parent population.

[0286] All safety analyses were conducted on the safety parent population defined as all registered subjects who received at least one administration.

[0287] Analysis of efficacy. The analysis of efficacy was performed on the mITT population and the EE population as described above.

[0288] Analysis of safety. The analysis of safety was performed based on all safety populations.

[0289] All adverse events (AEs) were coded into System Organ Class (SOC) and Preferred Term (PT) using the Medical Dictionary for Regulatory Activities (MedDRA®). The number of treatment-emergent adverse events (TEAEs), and the number of subjects who developed TEAEs (along with the percentage) were tabulated for each SOC and PT.

[0290] TEAE is an AE that occurred for the first time after the start of administration, or an AE whose severity worsened after the start of administration if it had occurred before administration.

[0291] Summaries are presented separately for the categories of TEAE, TEAE by severity, treatment-related TEAE, and serious TEAE.

[0292] When evaluating changes in safety parameters, the baseline is defined as the last measurement before the first administration.

[0293] Abnormalities in the evaluation of hematology, blood chemistry, and electrocardiogram are summarized.

[0294] HRCT is evaluated for the safety of the study at baseline and at the end of the study.

[0295] Biomarker analysis: Analyze bronchoalveolar lavage fluid (BAL) and / or blood samples to examine the relationship between biomarker concentration and clinical outcomes at each visit. In addition, evaluate the relationship between the biomarker and clinical outcomes to determine whether the biomarker can predict the study results. For details of the analysis, please refer to the SAP.

[0296] Example 7: Trial protocol for administration of MSC-NTF exosomes in a mouse model of lung injury

[0297] Background

[0298] Lung diseases targeted by EXO-MSC-NTF include adult respiratory distress syndrome (ARDS), interstitial pulmonary fibrosis (IPF), bronchopulmonary dysplasia (BPD), and chronic obstructive pulmonary disease (COPD).

[0299] Adult respiratory distress syndrome (ARDS) affects 150,000 people annually in the United States (16 per 100,000 population), and the acute-phase mortality rate is 30 - 70%. Potential benefits of EXO-MSC-NTF therapy include reduced mortality, shortened ICU or hospital stay, improved ventilation, and reduced need for ventilation support. ARDS is associated with shock, sepsis, pneumonia (including COVID-19), transfusion, gastric aspiration, and trauma.

[0300] Interstitial pulmonary fibrosis (IPF) affects 50,000 people annually in the United States (10 per 100,000 population), and the median survival period after diagnosis is 2 - 3 years. Potential benefits of EXO-MSC-NTF therapy include reduced mortality, shortened ICU or hospital stay, improved ventilation, reduced need for lung transplantation, and reduced need for ventilation support.

[0301] Bronchopulmonary dysplasia (BPD) is seen in 35% of births before 28 weeks of gestation, and approximately 18,000 infants are affected annually in the United States. The mortality rate of BPD is approximately 40 - 60% in infants with a birth weight of less than 1500 g. Potential benefits of EXO-MSC-NTF therapy include reduced mortality, shortened ICU or hospital stay, improved ventilation, improved lung development, and reduced need for ventilation support.

[0302] Chronic obstructive pulmonary disease (COPD) affects 15 million people annually in the United States (44.3 per 100,000 population). In severe COPD, the 5-year mortality rate is 40 - 70% and the 2-year mortality rate is 50%. Potential benefits of EXO-MSC-NTF therapy include reduced mortality, shortened ICU or hospital stay, improved ventilation, and reduced need for ventilation support.

[0303] Other animal models

[0304] Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in extremely preterm infants. BPD impairs lung development and causes severe respiratory complications over a long period, from childhood to adulthood. The understanding of BPD and the potential for developing treatment strategies have been obtained from large (baboon, sheep, pig) and small (rabbit, rat, mouse) animal models. These models are mainly aimed at inducing alveolar simplification similar to that seen in infants with BPD.

[0305] Various mouse models of BPD mainly focus on hyperoxia-induced lung injury. There are also hypoxia, hypoxia / hyperoxia, inflammation-induced models, and transgenic models.

[0306] Animal models of chronic obstructive pulmonary disease (COPD) are mainly induced in mice, guinea pigs, and rats. In most studies, this model is induced by exposure to cigarette smoke (CS), intratracheal lipopolysaccharide (LPS), and intranasal elastase. There are variations in the time course and dosage of the inducing factors used in the studies. The main measurement parameters were, in most studies, lung pathological data and lung inflammation (both inflammatory cells and inflammatory mediators), and in a very small number of published studies, tracheal reactivity (TR) (quoted in "Ghorani V, Boskabady MH, Khazdair MR, Kianmeher M. Experimental animal models for COPD: a methodological review. Tob Induc Dis. 2017 May 2;15:25", which is incorporated herein by reference).

[0307] EXO-MSC-NTF exerts unique effects, in part, through the paracrine secretion of vascular endothelial growth factor (VEGF), amphiregulin (AREG), and leukemia inhibitory factor (LIF).

[0308] VEGF is useful for the treatment of acute lung injury through its beneficial effects on type II alveolar epithelial cells. AREG regulates the recovery of the lungs and fibroblast function in mice after exposure to agricultural organic dust, and probably protects mice from LPS-induced acute lung injury by maintaining lung tissue homeostasis, suppressing TNF-α-induced alveolar epithelial cell death via EGFR signaling, and increasing the number of pathogenic memory T helper 2 cells that control airway fibrotic responses. LIF plays an important role in reducing chronic airway inflammation and protecting the lungs during viral pneumonia. Also, LIF is decreased by chronic smoking.

[0309] Purpose of the study

[0310] The purpose of this study is to examine the efficacy of bone marrow-derived mesenchymal stem cells (MSCs) and EXO-MSC-NTF (by intratracheal or aerosol inhalation) in a murine model of bleomycin, which is a murine model of another model of inflammation and fibrosis.

[0311] Bleomycin, a chemotherapeutic antibiotic produced by the bacterium "Streptomyces verticillus", is used as an agent that induces experimental pulmonary fibrosis. It mainly causes inflammatory and fibrotic reactions in a short period after intratracheal administration. First, the concentration of inflammatory cytokines increases, and then the expression of fibrosis-promoting markers and collagen accumulation increase, reaching a peak around day 14.

[0312] Stem cell-derived EVs have been tested in experimental models of lung injury such as asthma, ARDS, COPD, IPF, pneumonia, pulmonary hypertension, and silicosis models, and promising results have been obtained ("Cruz FF, Rocco PRM. Stem-cell extracellular vesicles and lung repair. Stem Cell Investig. 2017 21;4:78, incorporated herein by reference"). The common pathological conditions of these lung diseases include inflammation and fibrosis.

[0313] Compared with the control (PBS), improvement in all clinical parameters tested is expected in MSC, and enhancement of the effect is expected in EXO-MSC-NTF.

[0314] Design of the study

[0315] 1. Model: Mouse model of bleomycin-induced lung injury

[0316] C57bl mice were intratracheally administered a single dose of 3 U / kg bleomycin sulfate solution to induce lung injury.

[0317] 2. Administration

[0318] Intratracheal administration was performed during the inflammatory phase (day 1, day 5) or the fibrotic phase (day 7, day 10) to individually evaluate the effect of exosomes on inflammation and fibrosis.

[0319] In addition, for the initial evaluation of this route of administration (RoA), intranasal administration was performed on mice in one test group.

[0320] 3. Test groups

[0321]

Table 5

[0322] Analysis

[0323] · Oxygen saturation during the study period (4 - 5 time points). · Collection of BAL fluid and serum at the end of the study (measurement of inflammatory factors in BAL fluid and serum). · Histopathological examination of the lungs and quantification of fibrosis by the Ashcroft score (a score of pulmonary fibrosis ranging from 0 (normal lung) to 8) (“Ashcroft T, Simpson JM, Timbrell V (1988) Simple method of estimating severity of pulmonary fibrosis on a numerical scale. Journal of clinical pathology. 1988;41(4):467-70”; incorporated herein by reference). · Expression of fibrosis and cytokine mRNA panels in lung tissue (NanoString analysis). · Collagen content of lung tissue.

[0324] Results

[0325] As a result, favorable effects on oxygen saturation and body weight were confirmed in mice administered EXO-MSC and EXO-MSC-NTF intratracheally, compared to the control group.

[0326] A significant improvement in oxygen saturation was provided by EXO-MSC-NTF in mice administered, compared to the control, for both dosing schedules (day 1 and day 5 (Figure 16A), day 7 and day 10 (Figure 16B)), and for the dosing schedule of day 1 and day 5, EXO-MSC-NTF showed a superior effect compared to EXO-MSC. Administration of EXO-MSC-NTF by inhalation provided a significant benefit in oxygenation compared to the control (Figures 16C and 16F).

[0327] A significant improvement in weight gain was obtained only with EXO-MSC-NTF in the dosing schedule of administering on day 1 and day 5 (Figure 16D).

[0328] From the foregoing description of the specific embodiments, the general nature of the present invention should be sufficiently apparent. Thereby, others may, by applying current knowledge, without undue experimentation and without departing from the general concept, readily modify and / or adapt the above specific embodiments for various uses. Accordingly, such modifications and adaptations should be understood to be within the meaning and scope of the equivalents of the disclosed embodiments and are so intended. It is to be understood that the expressions and terms used herein are for the purpose of description and should not be regarded as limiting. Means, materials, and steps for performing the various disclosed functions may take various alternative forms without departing from the present invention.

Claims

1. A pharmaceutical composition for treating a lung disease or its symptoms in a patient in need of treatment, comprising: (a) a plurality of neurotrophic factor-secreting mesenchymal stem cells (MSC-NTF); (b) EXO-MSC-NTF defined as a plurality of small extracellular vesicles (sEV) derived from neurotrophic factor-secreting mesenchymal stem cells (MSC-NTF); and (c) a combination of neurotrophic factor-secreting mesenchymal stem cells (MSC-NTF) and EXO-MSC-NTF, wherein the active agent is selected from the group consisting of: the NTF includes vascular endothelial growth factor (VEGF), amphiregulin (AREG), and leukemia inhibitory factor (LIF), and the pharmaceutical composition is administered to the patient using a therapeutically effective regimen.

2. The pharmaceutical composition according to claim 1, wherein the lung disease includes a viral lung infection or a non-viral lung infection.

3. The pharmaceutical composition according to claim 1 or 2, wherein the active agent is neurotrophic factor-secreting mesenchymal stem cells (MSC-NTF).

4. The pharmaceutical composition according to claim 1 or 2, wherein the active agent is EXO-MSC-NTF.

5. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition contains about 10 ９ to about 10 １３ EXO-MSC-NTFs, and is a pharmaceutical composition.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition includes a combination of MSC-NTF and EXO-MSC-NTF.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the therapeutically effective regimen includes repeated administration of the active agent on different days, and the repeated administration includes administration on consecutive days or administration every other day.

8. The pharmaceutical composition according to claim 7, wherein the repeated administration includes administration on at least 5 different days.

9. The pharmaceutical composition according to claim 7, wherein the repeated administration includes administration on the 1st, 3rd, and 5th days.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition further includes an excipient.

11. The pharmaceutical composition according to claim 10, wherein the excipient includes PlasmaLyte A, DMEM, CryoStor® CS10 cryopreservation medium, or any combination thereof.

12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the volume of the pharmaceutical composition is from about 100 mL to about 120 mL.

13. A pharmaceutical composition according to any one of claims 1 to 12, wherein the administration of the pharmaceutical composition includes systemic administration, intravenous administration, intranasal administration, inhalation administration, intratracheal administration, direct injection, or any combination thereof.

14. A pharmaceutical composition according to any one of claims 1 to 13, wherein the symptoms are selected from the group consisting of pneumonia, acute respiratory distress syndrome (ARDS), interstitial pulmonary fibrosis (IPF), bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), multiple organ failure, fever, dry cough, fatigue, sputum production, loss of smell, shortness of breath, decreased oxygen saturation, muscle pain, joint pain, sore throat, headache, chills, nausea, vomiting, nasal congestion, diarrhea, inflammation, and fibrosis.

15. A pharmaceutical composition according to claim 14, wherein the symptoms are pneumonia, acute respiratory distress syndrome (ARDS), or a combination thereof.

16. A pharmaceutical composition according to any one of claims 2 to 15, wherein the viral lung infection is selected from the group consisting of coronavirus infection, severe acute respiratory syndrome (SARS) infection, Middle East respiratory syndrome (MERS) infection, influenza virus infection, Ebola virus infection, rabies virus infection, West Nile virus infection, dengue virus infection, respiratory syncytial virus (RSV) infection, and Zika virus infection.

17. A pharmaceutical composition according to claim 4, wherein the EXO-MSC-NTF, as compared to the corresponding EXO-MSC, (i) substantially contains less or does not contain at least one protein selected from the group consisting of A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q92598, P05023, and P62873, or A pharmaceutical composition substantially rich in at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

18. The pharmaceutical composition according to claim 17, wherein the EXO-MSC-NTF, as compared to the corresponding EXO-MSC, (i) substantially contains less at least one protein selected from the group consisting of A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q92598, P05023, and P62873, and (ii) substantially contains more at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

19. The pharmaceutical composition according to claim 17 or 18, wherein the EXO-MSC-NTF (i) contains 2.46 - 2.73 pg of LIF protein per 1 μg of total protein mass, (ii) contains 5.33 - 7.48 pg of AREG protein per 1 μg of total protein mass, (iii) contains 0.45 - 0.78 pg of HGF protein per 1 μg of total protein mass, or (iv) contains 0.027 - 0.065 pg of TSG6 protein per 1 μg of total protein mass.

20. The pharmaceutical composition according to claim 19, wherein the EXO-MSC-NTF (i) contains 2.46 - 2.73 pg LIF protein per 1 μg of total protein mass, (ii) contains 5.33 - 7.48 pg of AREG protein per 1 μg of total protein mass, (iii) contains 0.45 - 0.78 pg of HGF protein per 1 μg of total protein mass, and (iv) contains 0.027 - 0.065 pg of TSG6 protein per 1 μg of total protein mass.

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