Method and composition for treating pulmonary disease

Abstract

To provide a composition for treating a pulmonary disease.SOLUTION: A pharmaceutical composition for treating a pulmonary disease or symptoms thereof in a patient in need of treatment contains an active drug selected from the group consisting of: (a) a plurality of pluripotent mesenchymal stem cells (MSC) or a plurality of neurotrophic factor secretion mesenchymal stem cells (MSC-NTF); (b) EXO-MSC defined as a plurality of small extracellular vesicles (sEV) derived from the pluripotent mesenchymal stem cell, or EXO-MSC-NTF defined as a plurality of small extracellular vesicles (sEV) derived from the neurotrophic factor secretion mesenchymal stem cell (MSC-NTF); and (c) a combination of the pluripotent mesenchymal stem cell (MSC) or the neurotrophic factor secretion mesenchymal stem cell (MSC-NTF), and EXO-MSC or EXO-MSC-NTF, which is administered to the patient by using a therapeutically effective regimen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for treating pulmonary diseases. [Background technology]

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

[0003] Types of upper respiratory tract infections include the common cold (head cold), mild flu, tonsillitis, laryngitis, and sinus infections. The most common symptom of an upper respiratory tract infection is a cough. Lung infections can also cause nasal congestion, runny nose, sore throat, sneezing, muscle aches, and headaches.

[0004] Lower respiratory tract infections are found in the lungs and respiratory tract. They can be caused by viral infections, such as severe influenza, or bacterial infections, such as tuberculosis. Symptoms of lower respiratory tract infections include a severe cough that produces mucus (phlegm), shortness of breath, chest tightness, and wheezing when breathing out.

[0005] The COVID-19 pandemic, caused by SARS-CoV-2, causes mild, moderate, or severe illness, with severe clinical manifestations including pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and septic shock.

[0006] An as yet undetermined proportion of affected individuals experience sudden clinical deterioration approximately one week after onset, with rapidly progressive respiratory failure, multiple organ dysfunction (MOD), and multiple organ failure (MOF).

[0007] Concerns that COVID-19 will cause severe illness, respiratory failure, and death are at the heart of public anxiety. Acute respiratory distress syndrome (ARDS) caused by COVID-19 is associated with a mortality rate of over 50%. However, there are currently no effective curative strategies for treating ARDS, a type of respiratory failure accompanied by widespread inflammation and abnormal cytokine production, as evidenced by bronchoalveolar lavage (BAL) testing. Furthermore, despite recent advances and intensive care, ARDS is accompanied by severe systemic inflammation and multiple organ failure, posing an unacceptable compounding impact on existing medical resources. COVID-19 viral load has been demonstrated to correlate with lung function and outcomes, as well as inflammatory biomarkers, suggesting that therapies that inhibit viral replication may be synergistic with those that inhibit pulmonary inflammatory disease. Clearly, there is a strong need for therapies that can minimize the impact of COVID-19 on ARDS, sepsis, and multiple organ failure.

[0008] Furthermore, improved treatments and therapeutic strategies are needed to address the ongoing and future COVID-19 pandemic. Summary of the Invention [Means for solving the problem]

[0009] In one aspect, the present invention provides a method for treating a pulmonary viral infection or a symptom thereof in a patient in need thereof, the method comprising administering to the patient using a therapeutically effective regimen 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 neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs); (b) EXO-MSCs, which are defined as a plurality of small extracellular vesicles (sEVs) derived from pluripotent mesenchymal stem cells (MSCs), or EXO-MSC-NTFs, which are defined as a plurality of small extracellular vesicles (sEVs) derived from neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs) (NurOwn®); and (c) a combination of pluripotent mesenchymal stem cells (MSCs) or neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs) with EXO-MSCs or EXO-NSC-NTFs.

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

[0011] In some embodiments, the active agent is a multipotent mesenchymal stem cell (MSC). In some embodiments, the active agent is a combination of a multipotent mesenchymal stem cell (MSC) and an 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 contains about 5×10 6 ~About 300×10 6 Contains pluripotent mesenchymal stem cells (MSCs).

[0014] In some embodiments, the pharmaceutical composition contains about 15×10 6 ~About 100×10 6 Contains pluripotent mesenchymal stem cells (MSCs).

[0015] In some embodiments, the pharmaceutical composition contains about 15×106 ~About 20×10 6 Contains pluripotent mesenchymal stem cells (MSCs).

[0016] In some embodiments, the pharmaceutical composition contains about 80×10 6 ~About 100×10 6 Contains pluripotent mesenchymal stem cells (MSCs).

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

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

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

[0020] In some embodiments, the pharmaceutical composition contains about 3×10 10 ~Approx. 3×10 11 Contains EXO-MSCs or EXO-MSC-NTFs.

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

[0022] In some embodiments, the total amount of active agent administered to a patient is (a) Approximately 75 × 10 6 ~About 500×10 6 Multipotent mesenchymal stem cells (MSCs), (b) Approximately 5×10 11 EXO-MSCs or EXO-MSC-NTFs, and (c) Approximately 75×10 6 ~About 500×10 6 MSCs and approximately 5 x 1011 in combination with EXO-MSC or EXO-MSC-NTF, is selected from the group consisting of:

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

[0024] In some embodiments, a therapeutically effective regimen comprises a single administration of an active agent. In some embodiments, a therapeutically effective regimen comprises multiple administrations of an active agent.

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

[0026] In some embodiments, the repeated administration comprises administration on at least five different days.

[0027] In some embodiments, the multiple administration comprises administration on consecutive days.

[0028] In some embodiments, the repeated administration comprises administration 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 freezing medium.

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

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

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

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

[0037] In some embodiments, the methods of the present disclosure comprise inhaled administration of the pharmaceutical composition.

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

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

[0040] In some embodiments, the methods of the present disclosure comprise administering the pharmaceutical composition by inhalation.

[0041] In some embodiments, the symptom is 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 condition is acute respiratory distress syndrome (ARDS).

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

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

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

[0047] [Figure 1] Histopathological micrographs of lung lesions in mice. [Figure 2A] Score for alveolar wall thickening (Figure 2A). [Figure 2B] Total score in the trachea (Figure 2B). EV = EXO-MSC. [Figure 3A] Serum concentrations of cytokines: IL-1β serum (Fig. 3A); [Figure 3B] IL-6 serum (Figure 3B). [Figure 3C] MCP-1 serum (Fig. 3C). [Figure 3D] IFN-γ serum (Figure 3D). [Figure 3E] TNF-α serum (Fig. 3E). [Figure 4A] Lung fluid (bronchoalveolar fluid, BAL) concentrations of cytokines. *p≦0.05; **p≦0.01; ***p≦0.001. IL-1β BAL (Figure 4A). [Figure 4B] IL-6 BAL (Fig. 4B). [Figure 4C] IP-10 BAL (Figure 4C). [Figure 4D] IFN-γ BAL (Fig. 4D ). [Figure 4E] TNF-α BAL (Fig. 4E). [Figure 4F] MCP-1 BAL ( Figure 4F ). [Figure 4G] IL-1α BAL (Fig. 4G). [Figure 5] Neutrophil blood levels. [Figure 6] Total severity score for acute lung injury following intratracheal administration: ap<0.05 no LPS control; bp<0.05 LPS+Plasmalyte. [Figure 7A] Fibrin deposition score ap<0.05 no LPS control; cp≦0.01 LPS+Plasmalyte. [Figure 7B] Alveolar wall thickness score ap<0.05 no LPS control; cp≦0.01 LPS+Plasmalyte. [Figure 7C] Neutrophil score. [Figure 7D] Neutrophil counts in lung sections: ap<0.05 no LPS control; bp<0.05 LPS+Plasmalyte. [Figure 8] Representative histopathological micrographs of lung lesions. (A) Moderately affected lung: LPSEXO-MSC. (B) Moderately affected lung: LPSEXO-MSC-NTF. (C) Moderately to severely affected lung: LPS control (Plasmalyte). (D) Unaffected lung - healthy control (Plasmalyte). [Figure 9] Oxygen saturation (%). *p<0.05, **p<0.01, ***p<0.001 LPS+Plasmalyte group. [Figure 10A] Cytokine concentrations in bronchoalveolar fluid. Figure 10A: IFN-γ concentration bp<0.05 LPS+Plasmalyte. [Figure 10B] FIG. 10B: Concentration of IL-6 ap<0.05 no LPS control, bp<0.05 LPS+Plasmalyte. [Figure 11A] Cytokine concentrations in bronchoalveolar fluid. Figure 11A: IL-10 concentration cp ≤ 0.01 LPS + Plasmalyte. [Figure 11B] Figure 11B: Concentrations of RANTES. ap<0.05 no LPS control, bp<0.05 LPS plasmalite. [Figure 12]Cytokine concentrations in bronchoalveolar fluid. TNF-α concentration: ap<0.05 no LPS control, bp<0.05 LPS+Plasmalyte, b#p=0.058 LPS+Plasmalyte. [Figure 13A] Immunomodulatory activity of sEVs as determined by inhibition of IFN-γ (FIG. 13A) and TNF-α (FIG. 13B) secretion by activated PBMCs. Cell culture supernatant ELISA was performed after incubation with EXO-MSCs or EXOMSC-NTFs from four independent donors compared to untreated activated PBMCs. Mean ± SEM, *p<0.05 paired t-test. [Figure 13B] Immunomodulatory activity of sEVs as determined by inhibition of IFN-γ (FIG. 13A) and TNF-α (FIG. 13B) secretion by activated PBMCs. Cell culture supernatant ELISA was performed after incubation with EXO-MSCs or EXOMSC-NTFs from four independent donors compared to untreated activated PBMCs. Mean ± SEM, *p<0.05 paired t-test. [Figure 14A] Differences in protein cargo between EXO-MSC-NTFs and EXO-MSCs. [Figure 14B] ELISA of EXO-MSC and EXO-MSC-NTF lysates from three independent donors showed higher concentrations of LIF (FIG. 14A) and AREG (FIG. 14B) in EXO-MSC-NTFs. [Figure 14C] HGF (FIG. 14C) and TSG-6 (FIG. 14D) were detected in both EXO-MSCs and EXO-MSC-NTFs, but there was no significant difference. [Figure 14D] Mean ± SEM, n = 3, *p < 0.05 paired t test. [Figure 15] Schematic diagram showing the timeline of one embodiment of the methods provided by the present invention: Five daily doses (Days 1, 2, 3, 4, 5) or three alternate-day doses (Days 1, 3, 5) with a screening visit / baseline assessment and follow-up assessments up to Day 28. [Figure 16A]FIG. 1 is a schematic diagram showing the administration of EXO-MSCs, EXO-MSC-NTFs, or PBS as a control on bleomycin sulfate-induced lung injury in mice. [Figure 16B] Administration was performed during the inflammatory phase (days 1 and 5, Figures 16A and 16D) or the fibrotic phase (days 7 and 10, Figures 16B and 16E), and the effects of exosomes on inflammation and fibrosis were evaluated separately. [Figure 16C] One test group was administered by inhalation (FIGS. 16C and 16F) and compared to two PBS controls (days 1, 5 and 7, 10). [Figure 16D] Results are presented as mean ± SEM. [Figure 16E] 16A, 16B, and 16C show oxygen saturation. [Figure 16F] Figures 16D, 16E, and 16F show weight. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides methods, compositions, and treatment regimens for treating a variety of human diseases commonly known as "pulmonary viral infections" or "respiratory viral infections."

[0049] In accordance with the principles of the present invention, therapeutic agents are administered to a patient at predetermined doses and according to predetermined treatment regimens to maximize the therapeutic effect of the therapeutic agent while minimizing inconvenience and risk to the patient receiving the treatment.

[0050] As one of ordinary skill in the art will appreciate, different therapeutic agents, administered in different doses and by different modes of administration, will produce different therapeutic results.

[0051] In one aspect, the present invention provides a method for treating a pulmonary viral infection or a symptom thereof in a patient in need thereof, the method comprising administering to the patient using a therapeutically effective regimen 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 neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs); (b) EXO-MSCs, which are defined as a plurality of small extracellular vesicles (sEVs) derived from pluripotent mesenchymal stem cells (MSCs), or EXO-MSC-NTFs, which are defined as a plurality of small extracellular vesicles (sEVs) derived from neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs) (NurOwn®); and (c) a combination of pluripotent mesenchymal stem cells (MSCs) or neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs) with EXO-MSCs or EXO-NSC-NTFs.

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

[0053] In some embodiments, the active agent is pluripotent mesenchymal stem cells (MSCs). In some embodiments, the MSCs are administered to the patient at least two times. In some embodiments, the MSCs are administered to the patient at least three times. In some embodiments, the MSCs are administered to the patient at least four times. In some embodiments, the MSCs are administered to the patient at least five times. In some embodiments, the MSCs are administered to the patient two times. In some embodiments, the MSCs are administered to the patient three times. In some embodiments, the MSCs are administered to the patient four times. In some embodiments, the MSCs are administered to the patient five times. In some embodiments, the MSCs are administered to the patient no more than two times. In some embodiments, the MSCs are administered to the patient no more than three times. In some embodiments, the MSCs are administered to the patient no more than four times. In some embodiments, the MSCs are administered to the patient no more than five times. In some embodiments, the MSCs are administered to the patient 1 to 5 times. In some embodiments, the MSCs are administered to the patient 2 to 5 times. In some embodiments, the MSCs are administered to the patient 3 to 5 times, hi some embodiments, the MSCs are administered to the patient 4 to 5 times.

[0054] In some embodiments, the MSCs are administered on days 1, 3, and 5. In some embodiments, the MSCs are administered on days 1, 3, 5, 7, and 9.

[0055] In some embodiments, the active agent is a combination of MSCs and EXO-MSCs. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient at least two times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient at least three times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient at least four times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient at least five times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient two times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient three times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient four times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient five times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient no more than two times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient three or fewer times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient four or fewer times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient five or fewer times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient one to five times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient two to five times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient three to five times. In some embodiments, the combination of MSCs and EXO-MSCs is administered to the patient four to five times.

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

[0057] In some embodiments, the pharmaceutical composition contains about 1×105 ~About 1000×10 7 In some embodiments, the pharmaceutical composition comprises about 5 x 10 MSCs. 5 ~About 300×10 7 In some embodiments, the pharmaceutical composition comprises about 1 x 10 MSCs. 6 ~About 1000×10 6 In some embodiments, the pharmaceutical composition comprises about 5 x 10 MSCs. 6 ~About 300×10 6 Includes MSCs.

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

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

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

[0061] In some embodiments, the active agent is EXO-MSCs. In some embodiments, the EXO-MSCs are administered to the patient at least two times. In some embodiments, the EXO-MSCs are administered to the patient at least three times. In some embodiments, the EXO-MSCs are administered to the patient at least four times. In some embodiments, the EXO-MSCs are administered to the patient at least five times. In some embodiments, the EXO-MSCs are administered to the patient two times. In some embodiments, the EXO-MSCs are administered to the patient three times. In some embodiments, the EXO-MSCs are administered to the patient four times. In some embodiments, the EXO-MSCs are administered to the patient five times. In some embodiments, the EXO-MSCs are administered to the patient no more than two times. In some embodiments, the EXO-MSCs are administered to the patient no more than three times. In some embodiments, the EXO-MSCs are administered to the patient no more than four times. In some embodiments, the EXO-MSCs are administered to the patient no more than five times. In some embodiments, EXO-MSCs are administered to the patient 1 to 5 times. In some embodiments, EXO-MSCs are administered to the patient 2 to 5 times. In some embodiments, EXO-MSCs are administered to the patient 3 to 5 times. In some embodiments, EXO-MSCs are administered to the patient 4 to 5 times.

[0062] In some embodiments, EXO-MSCs are administered on days 1, 3, and 5. In some embodiments, EXO-MSCs are administered on days 1, 3, 5, 7, and 9.

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

[0064] In some embodiments, EXO-MSC-NTFs are administered on days 1, 3, and 5. In some embodiments, EXO-MSC-NTFs are administered on days 1, 3, 5, 7, and 9.

[0065] In some embodiments, the pharmaceutical composition comprises about 10 9 ~about 10 13 In some embodiments, the pharmaceutical composition comprises about 3 x 10 EXO-MSCs or EXO-MSC-NTFs. 9 ~Approx. 3×10 12In some embodiments, the pharmaceutical composition comprises about 10 EXO-MSCs or EXO-MSC-NTFs. 9 ~about 10 12 In some embodiments, the pharmaceutical composition comprises about 10 EXO-MSCs or EXO-MSC-NTFs. 10 ~about 10 12 Contains EXO-MSCs or EXO-MSC-NTFs.

[0066] In some embodiments, the pharmaceutical composition contains about 2×10 9 ~Approx. 5×10 11 In some embodiments, the pharmaceutical composition comprises about 3 x 10 EXO-MSCs or EXO-MSC-NTFs. 9 ~Approx. 3×10 11 In some embodiments, the pharmaceutical composition comprises about 5 x 10 EXO-MSCs or EXO-MSC-NTFs. 9 ~Approx. 2×10 11 Contains EXO-MSCs or EXO-MSC-NTFs.

[0067] In some embodiments, the pharmaceutical composition contains about 2×10 10 ~Approx. 5×10 11 In some embodiments, the pharmaceutical composition comprises about 3 x 10 EXO-MSCs or EXO-MSC-NTFs. 10 ~Approx. 3×10 11 In some embodiments, the pharmaceutical composition comprises about 5 x 10 EXO-MSCs or EXO-MSC-NTFs. 10 ~Approx. 2×10 11 Contains EXO-MSCs or EXO-MSC-NTFs.

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

[0069] In some embodiments, the total amount of active agent administered to a patient is (a) about 75×10 5 ~About 500×10 7 (b) approximately 5 × 10 MSCs 10 ~Approx. 5×10 12 EXO-MSCs or EXO-MSC-NTFs, and (c) approximately 75 × 10 5 ~About 500×10 7 MSCs and approximately 5 x 10 9 ~Approx. 5×10 12 a combination of EXO-MSCs or EXO-MSC-NTFs.

[0070] In some embodiments, the total amount of active agent administered to a patient is (a) about 75×10 5 ~About 500×10 7 (b) approximately 5 × 10 MSCs 10 ~Approx. 5×10 12 EXO-MSCs or EXO-MSC-NTFs, and (c) approximately 75 × 10 5 ~About 500×10 7 MSCs and approximately 5 x 10 10 ~Approx. 5×10 12 a combination of EXO-MSCs or EXO-MSC-NTFs.

[0071] In some embodiments, the total amount of active agent administered to a patient is (a) about 15×10 6 ~Approx. 250×10 7 (b) approximately 1 × 10 MSCs 11 ~Approx. 25×10 11 EXO-MSCs or EXO-MSC-NTFs, and (c) approximately 15 × 10 6 ~Approx. 250×10 7 MSCs and approximately 1 x 10 11 ~Approx. 25×10 11 one EXO-MSC or a combination with EXO-MSC-NTF.

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

[0073] In some embodiments, the total amount of active agent administered to a patient is (a) about 30×10 6 ~About 100×10 7 (b) approximately 2.5 × 10 MSCs 11 ~Approx. 1×10 12 EXO-MSCs or EXO-MSC-NTFs, and (c) approximately 30 × 10 6 ~About 100×10 7 MSCs and approximately 2.5 x 10 11 ~Approx. 1×10 12 a combination of EXO-MSCs or EXO-MSC-NTFs.

[0074] In some embodiments, the total amount of active agent administered to a patient is (a) about 75×10 6 ~About 500×10 6 (b) approximately 5 × 10 MSCs 11 EXO-MSCs or EXO-MSC-NTFs, and (c) approximately 75 × 10 6 ~About 500×10 6 MSCs and approximately 5 x 10 11 a combination of EXO-MSCs or EXO-MSC-NTFs.

[0075] In some embodiments, the MSCs comprise 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 in different months. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent in different weeks. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent on different days. In some embodiments, a therapeutically effective regimen comprises repeated administration of an active agent at different times on the same day.

[0077] In some embodiments, the repeated administration comprises administration on at least two different days. In some embodiments, the repeated administration comprises administration on at least three different days. In some embodiments, the repeated administration comprises administration on at least four different days. In some embodiments, the repeated administration comprises administration on at least five different days.

[0078] In some embodiments, the repeated administration comprises administration on consecutive days. In some embodiments, the repeated administration comprises administration on at least two consecutive days. In some embodiments, the repeated administration comprises administration on at least three consecutive days. In some embodiments, the repeated administration comprises administration on at least four consecutive days. In some embodiments, the repeated administration comprises administration on at least five consecutive days.

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

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

[0081] In some embodiments, the volume of the pharmaceutical composition is 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 comprise systemic administration of the pharmaceutical composition. In some embodiments, the methods of the present disclosure comprise intravenous administration of the pharmaceutical composition. In some embodiments, the methods of the present disclosure comprise 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 has been frozen and then thawed. 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 has been frozen and then thawed.

[0084] In some embodiments, the symptom is 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 loss of smell. In some embodiments, the symptom is shortness of breath. In some embodiments, the symptom is muscle pain. In some embodiments, the symptom is joint pain. In some embodiments, the symptom is sore throat. 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 pulmonary infection is selected from the group consisting of a coronavirus infection, a severe acute respiratory syndrome (SARS) infection, a Middle East respiratory syndrome (MERS) infection, an influenza virus infection, an Ebola virus infection, a rabies infection, a West Nile virus infection, a dengue virus infection, a respiratory syncytial virus (RSV) infection, and a Zika virus infection.

[0087] In some embodiments, the viral pulmonary infection is a coronavirus infection. In some embodiments, the viral pulmonary infection is a severe acute respiratory syndrome (SARS) infection. In some embodiments, the viral pulmonary infection is a Middle East respiratory syndrome (MERS) infection. In some embodiments, the viral pulmonary infection is an influenza virus infection. In some embodiments, the viral pulmonary infection is an Ebola virus infection. In some embodiments, the viral pulmonary infection is a rabies infection. In some embodiments, the viral pulmonary infection is a West Nile virus infection. In some embodiments, the viral pulmonary infection is a dengue virus infection. In some embodiments, the viral pulmonary infection is a respiratory syncytial virus (RSV) infection. In some embodiments, the viral pulmonary 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 MSCs and EXO-MSCs. In some embodiments, the active agent is a combination of MSC-NTFs and EXO-MSC-NTFs.

[0090] In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient at least four times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient at least five times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient two times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient three times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient four times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient five times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient two or fewer times. In some embodiments, the combination of MSC-NTFs and EXO-MSC-NTFs is administered to the patient three or fewer times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient four or fewer times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient five or fewer times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient one to five times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient two to five times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient three to five times. In some embodiments, the combination of MSC-NTF and EXO-MSC-NTF is administered to the patient four to five times.

[0091] In some embodiments, the EXO-MSC-NTFs have, compared to their corresponding EXO-MSCs, (i) A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q9259 8, P05023, and P62873, or (ii) substantially enriched in 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, the EXO-MSC-NTFs have, compared to their corresponding EXO-MSCs, (i) A1L4H1, P49747, P02452, Q7Z304, Q5VTE0, P68104, Q05639, P60903, P08123, P09619, Q15113, P15144, O43854, Q71U36, P0DPH8, P0DPH7, Q6PEY2, Q925 98, P05023, and P62873, and (ii) substantially enriched in 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, the EXO-MSC-NTFs contain (i) 2.46 to 2.73 pg of LIF protein per μg of total protein, (ii) 5.33 to 7.48 pg of AREG protein per μg of total protein, (iii) 0.45 to 0.78 pg of HGF protein per μg of total protein, or (iv) 0.027 to 0.065 pg of TSG6 protein per μg of total protein.

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

[0095] The terms "mesenchymal stem cells," "mesenchymal stromal cells," "multipotent stromal cells," "MSCs," or "MSCs" are used interchangeably to refer to adult cells that are not terminally differentiated, that can divide to generate stem cells, or that can irreversibly differentiate to generate cells of mesenchymal lineages or transdifferentiate into cells of other non-mesodermal lineages, such as neural lineages.

[0096] The source of the MSCs may be a healthy subject, the subject to be treated, a donor that is immunologically matched to the subject to be treated, or a donor that is immunologically mismatched. In some embodiments, the source of the MSCs may be a subject suffering from a neurodegenerative disease. In some embodiments, the MSCs comprise autologous cells. In other embodiments, the MSCs comprise allogeneic cells. As exemplified herein, EXO-MSCs and EXO-MSC-NTFs express few MHC-I and MHC-II molecules, potentially making immunological matching between exosomes and a human recipient redundant.

[0097] Mesenchymal stem cells (MSCs) are found in almost all tissues and can be isolated from a variety of tissues. While bone marrow (BM) is the most widely recognized source of MSCs, recent studies have revealed additional sources of MSCs, 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 present in virtually all vascular tissues throughout the body.

[0098] In some embodiments, the MSCs described herein are isolated from any tissue they are present in, including, but 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, adipocyte MSCs, dental pulp MSCs, placental MSCs, synovial MSCs, peripheral blood MSCs, oral mucosa MSCs, periodontal ligament MSCs, endometrial MSCs, umbilical Wharton's jelly MSCs, and umbilical cord blood MSCs.

[0100] The term "extracellular vesicles" (EVs) refers to a heterogeneous population of endoplasmic reticulum of cellular origin, derived from endosomal compartments (exosomes) or resulting from shedding from the plasma membrane. Extracellular vesicles (EVs) are membrane-enclosed nanoscale particles released from essentially all prokaryotic and eukaryotic cells. EV diameters range from approximately the size of the smallest physically possible unilamellar liposomes (approximately 20–30 nanometers) to over 10 micrometers, with the majority of EVs being less than 200 nm. EVs are defined as exosomes, microvesicles, or apoptotic bodies based on their size and synthesis pathway. They transport cargo from parent cells, including proteins, nucleic acids, lipids, metabolites, and even organelles. Exosomes are small EVs (ranging from 30–150 nm) generated by invagination of the endosomal membrane to form 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 embodiments, 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 further comprises one or more miRNA molecules selected from the group consisting of 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. The population lacks or is a combination of 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.

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

[0103] The above-described embodiments are intended to be illustrative only. Those skilled in the art will appreciate that various changes, modifications, and variations may be made to the specific embodiments. The claims should not be limited to the specific embodiments described herein, but should be construed in a manner consistent with the specification as a whole.

[0104] Example

[0105] Example 1: Bone marrow harvest for isolation of mesenchymal stem cells (MSCs)

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

[0107] Prior to the bone marrow aspiration (BMA) procedure, documentation reporting donor test results for HIV1, HIV2, HBV, HCV, HTLV, syphilis, and COVID-19 was submitted. Positive test results that would exclude the BMA 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 BMA procedure.

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

[0109] Example 2: Expansion of MSCs

[0110] The first step in the manufacturing process involves the isolation of mononuclear cells (MNC) from whole bone marrow by Ficoll density gradient centrifugation.

[0111] hMSCs were enriched from mononuclear cells (MNCs) in vitro (in a Corning two-chamber cell stack) by utilizing their ability to adhere to plastic. To prevent potential risks of infection or host immune response, the production process was performed in xeno-free growth medium containing 10% human platelet lysate (PL) and the specified growth medium (PM). For the first 16–24 h, cells were seeded onto the PM in a two-chamber cell stack (tissue culture vessel) at 37°C / 5% CO2. During this stage, MSCs that adhered to the plastic adhered to the surface of the cell stack, while non-adherent mononuclear cells floated in the supernatant. The PM was replaced with fresh PM (P0). During passage 0, the hMSC medium was replaced 4–6 times. After a maximum of 15 days, P0 MSCs were harvested and cryopreserved.

[0112] After harvesting 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 expression of CD14, CD34, CD45, and HLA-DR (<5%) as determined by flow cytometry. MSCs comply with the specifications.

[0114] 15×10 6 Eighteen cryotubes containing 100 cells / tube were frozen 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.

[0115] After cryopreservation of P0 MSCs, the cells were thawed and seeded for expansion (passage 1: P1). Thawed hMSCs were seeded at 1,000 cells / cm in proliferation medium (PM) in a two-chamber cell stack. 2 The cells were seeded at a concentration of 0.01% for 7–8 days. Growth medium (PM) was changed every 3–4 days. After a maximum of 7–8 days, P1 cells were harvested and optionally cryopreserved (passage 1).

[0116] After harvesting of passage 1 cells, MSC cultures were sampled for in-process sterility and mycoplasma testing prior to cryopreservation.

[0117] 25×10 6 Cryotubes containing 100 cells / tube were cryopreserved in the vapor phase of a liquid nitrogen freezer (-196 °C), which provides stable cryogenic storage.

[0118] After cryopreservation of MSCs at P1, the cells were thawed and seeded for expansion (passage 2: P2). Thawed hMSCs were seeded at 1,000 cells / cm in Proliferation Medium (PM) in a two-chamber cell stack. 2 The cells were seeded at a concentration of 1000 for 7–8 days. The growth medium (PM) was changed every 3–4 days. After a maximum of 7–8 days, P2 cells were harvested and cryopreserved (passage 2).

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

[0120] 30×10 6 Cryotubes containing 100 x 10 cells / tube were frozen and stored in the vapor phase of a liquid nitrogen freezer (-196°C), which provides stable cryogenic storage. 6 Five cryotubes are required to produce a single low dose (20 x 10 cells) for one patient. 6 One cryotube is required to produce 130 x 10 cells. 6Cryotubes containing 10 cells / tube are cryopreserved in the vapor phase of a liquid nitrogen freezer (-196 °C), which provides stable cryogenic storage.

[0121] For patient treatment with allogeneic MSCs, cells were thawed to create the final product. After thawing the MSCs, the cells were pooled, washed, and counted. The MSCs were then loaded into syringes and labeled. The MSC suspension was sampled for final bulk safety testing for sterility, Gram stain, and endotoxin. Alternatively, 130 × 10 6 Thaw cells from the cryotubes containing 100 cells / tube and immediately administer to the patient. Visually inspect the final syringe to ensure it meets specifications (the syringe is intact, the cell suspension is a turbid yellowish color, and there are virtually no visible particulates in the cell suspension).

[0122] Example 3: Production, purification, and characterization of EXO-MSCs, including EXO-MSC-NTFs

[0123] To scale up exosome production and improve yield, thawed MSCs (P0 or P1; see Example 2) were resuspended in growth medium (PM) and either seeded directly into a Quantum Cell Expansion System Bioreactor (Terumo BCT) or seeded into cell stacks for reseeding in a PBS Bioreactor System (PBS Biotech) and grown for several days.

[0124] Quantum

[0125] The Quantum Cell Expansion System is a functionally closed, automated hollow fiber bioreactor system. The bioreactor itself consists of approximately 11,500 hollow fibers, with a total intracapillary (IC) surface area of ​​2.1 m. 2The fluid circuit of the Quantum system is designed around two fluid loops: one intracapillary (IC) loop and one extracapillary (EC) loop in the hollow fiber.

[0126] The PBS Bioreactor System (PBS Biotech) is a vertical wheel, single-use bioreactor capable of providing uniform, low-shear, and scalable mixing over a wide range of work volumes.

[0127] Exosome bioreactor cell culture

[0128] The first step of the manufacturing process involves the isolation of mononuclear cells (MNCs) from whole bone marrow using Sepax2 (Cytiva), a fully automated, closed, and compact solution for the isolation of MNCs by Ficoll density gradient centrifugation. The Quantum Cell Expansion System was seeded with either Sepax-separated MNCs, thawed P0 MSCs, or P1 MSCs expanded in CellSTACK.

[0129] Prior to cell seeding, the bioreactor was coated with 5–10 mg of fibronectin for at least 4 hours to overnight using the "Coat Bioreactor Task." After 4 hours to overnight of bioreactor coating, excess fibronectin was washed off the bioreactor set, and the PBS solution was replaced with PM growth medium in DMEM without the addition of antibiotics / antimycotics by introducing cell culture medium into the bioreactor set using the IC / EC Washout Task.

[0130] Human MSCs (hMSCs) were enriched in vitro from mononuclear cells (MNCs) in a Quantum bioreactor by utilizing their ability to adhere to the surface of hollow fibers. To prevent potential risks of infection or host immune responses, the process was performed in xeno-free growth medium containing 10% human platelet lysate (PL without antibiotics or antimycotics) and the designated growth medium (PM). For the first 16–24 hours, cells were seeded in the growth medium (PM) within the Quantum system at 37°C / 5% CO2. During this phase, MSCs attached to the hollow fibers adhere to the bioreactor surface, while non-adherent mononuclear cells float in the supernatant. The PM was replaced with fresh PM (P0). After up to 15 days, the P0 MSCs were harvested, a portion cryopreserved (P0), and the remaining portion reseeded into a new Quantum Cell Expansion 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 brought up to 100 mL with growth medium (PM). The bag was then sterilely connected to the Quantum system's cell inlet line, and the cells were loaded into the fibronectin-coated IC side 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 expanded 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, MSCs (passage 1: P1) were harvested.

[0132] After harvesting P1 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.

[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, these cells must lack expression of CD14, CD34, CD45, and HLA-DR as determined by flow cytometry.

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

[0135] After cryopreservation of MSCs in P1, the cells were thawed and seeded for expansion (P2). Twenty million thawed and washed MSCs (cells were frozen and cryopreserved in liquid N2 at passages 0–2) were transferred to a cell infusion bag, and the total volume of the bag was brought up to 100 mL with 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-coated 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 expanded for 6–7 days using the cell feeding task, which adjusted the IC inflow rate according to the glucose consumption and lactate production rates 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 maximize the number of exosomes produced from the same cells, taking into account the glucose consumption and lactate production rates, the exosome-containing cell medium was collected 2–5 times every 48 h.

[0136] PBS3 MAG: A PBS3 MAG bioreactor equipped with a 3-liter single-use vessel (PBS Biotech) was loaded with 100–200 g of Synthemax II low-density microcarriers (Corning) or enhanced-attachment microcarriers (Corning). The vessel was then 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 allowed to equilibrate overnight.

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

[0138] EXO-MSCs were generated by culturing MSCs under continuous agitation for 5–7 days, maintaining a final PL concentration of 10% and performing 50–80% medium changes daily from day 3. On the final day of culture, the medium was replaced with platelet lysate-free medium for exosome harvesting. Cell culture media containing exosomes was harvested either at the end of the production process or every 48 h for a total of 2–5 times.

[0139] After collection, all collection media were pooled and exosomes were isolated.

[0140] EXO-MSC-NTFs were generated by culturing the cells under continuous agitation for 5–7 days, maintaining a final PL concentration of 10% and performing 50–80% medium changes daily from day 3. On the final day of culture, the growth medium was replaced with S2M differentiation medium (Dulbecco's modified Eagle's 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, supplemented with 200 μM ascorbic acid) for exosome collection after 72 hours. The exosome isolation steps were the same for EXO-MSCs and EXO-MSC-NTFs, as follows: EXO-MSCs and EXO-MSC-NTFs were isolated and purified using tangential flow filtration (TFF). To remove microcarriers, the exosome-containing medium was first passed through a 100 μm separation bag, and then the medium was filtered through a 0.8-1.2 μm filter to remove cellular debris. The exosome-containing filtrate was 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 to 5,000 cm 2A molecular weight cut-off (MWCO) membrane with a filtration area of ​​100 kDa (Repligen) was used. The exosome-containing sample was continuously pumped through the membrane system and recirculated. Small molecules, including free proteins not contained within or bound to membrane vesicles, passed through the membrane pores and were eluted as the filtrate, which was ultimately discarded. Molecules too large to pass through the membrane pores, such as exosomes (or larger microvesicles), continued to circulate as the retentate. To further remove contaminants smaller than the MWCO membrane's kDa, the sample was subjected to diafiltration at 5-10 times the volume. At the final filtration cycle, the sample was reduced to a volume of approximately 100 ml. Finally, the sample was sterilized using a 0.2 μm filter.

[0142] Nanoparticle tracking analysis

[0143] The amount and size of particles were measured using ZetaView Nanoparticle Tracking Analysis (ParticleMetrix), a laser scattering video microscope that tracks the movement of individual nanoparticles under Brownian motion. Five exposures were recorded for each sample at 11 measurement locations. Particle size was calculated according to the Stokes-Einstein equation using ZetaView software (ZetaView 8.02.28).

[0144] FACS analysis

[0145] Exosome phenotyping was performed using the MACSPlex Exosome Kit, which allows for the detection of 37 exosome surface epitopes and two 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 BM-MSC-derived exosomes revealed high expression of tetraspanins (a series of conserved proteins expressed on exosomes, including CD81, CD63, and CD9), MSC CD markers (CD44, CD29, and CD49e), and negative expression of hematopoietic CD markers (including CD4 and CD19), HLA-DR, and HLA-ABC.

[0146] Example 4: Evaluation of the efficacy of administration of EXO-MSCs in an ARDS mouse model

[0147] The purpose of this study is to examine 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 leading cause of coronavirus-related deaths.

[0148] The use of animal ARDS models allows us to investigate the efficacy of EXO-MSCs in suppressing clinical symptoms caused by the inflammatory response, thereby enabling the development of this treatment for ARDS. The LPS-induced ARDS model is an accepted model for severe human acute respiratory disease caused by coronavirus infection.

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

[0150] Animals; Choice of 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, orally intubated with a sterile plastic catheter, and intratracheally administered 800 μg of LPS dissolved in 50 μL of standard PBS. Naive mice (no LPS administration, test group 6) were injected with the same volume of PBS as controls.

[0153] Administration: Daily administration of EXO-MSCs via an endotracheal tube or 2.0 × 10 10 Intravenous administration was initiated at a concentration of 1 vesicles / 1 ml, 3 hours after LPS administration.

[0154] Sample collection: Blood was collected for whole blood hematology for cell counts and serum analysis. Total bronchoalveolar lavage (BAL) fluid was measured for T lymphocytes, B lymphocytes, eosinophils, neutrophils, dendritic cells, and monocytes / macrophages, as well as differential cell counts by FACS. BAL fluid samples were also analyzed for the presence of inflammatory cytokines. Lungs were removed from all animals sacrificed on day 3 for histopathological H&E.

[0155] Histological Assessment: Quantitative analysis of acute lung injury (ALI) was performed using a severity scoring scale of 0 to 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 to 5 neutrophils - score 1; more than 5 neutrophils - score 2. 2. Fibrin: not visible in the field - score 0; single neat band of fibrin in the air space - score 1; multiple eosinophil membranes - score 2. 3. Thickening of the alveolar walls: due to technical artifacts, only septal thickening more than twice the normal was considered. Less than twice the normal - score 0; 2 to 4 times the normal - score 1; more than 4 times the normal - score 2.

[0157] Figure 1 shows the histopathological results of the lungs of representative mice from test groups 1 to 6 (see Table 1).

[0158] Figure 2A shows the group scores for alveolar wall thickening based on histopathological results when exosomes were administered intratracheally to test group 1 ("No EVs" = "LPS + Plasmalyte") and test group 2 (EVs = EXO-MSCs). A statistically significant difference was observed, demonstrating that exosome therapy was effective in reducing alveolar wall thickening. Figure 2B shows the total acute lung injury score based on histopathological results when exosomes were administered intratracheally to test group 1 ("No EVs" = "LPS + Plasmalyte") and test group 2 (EVs = EXO-MSCs). A statistically significant difference was observed, demonstrating that exosome therapy was 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), and TNF-α (Figure 3E) in mice intratracheally administered with EXO-MSCs and mice administered with Plasmalyte. Statistically significant differences were observed ( * p<0.05), demonstrating that exosome therapy is effective in reducing serum cytokine concentrations.

[0160] Figure 4 shows the lung fluid 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 mice treated with EXO-MSCs intratracheally and mice treated with Plasmalyte. Statistically significant differences were observed, demonstrating that exosome therapy is effective in reducing lung fluid concentrations of cytokines.

[0161] Figure 5 shows the blood neutrophil concentrations in healthy animals (control group), the LPS control group, and the LPS + EXO-MSC-IV administration group. A statistically significant increase in blood neutrophil concentrations was observed in the LPS control group, whereas 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 efficacy of bone marrow-derived mesenchymal stem cells or EXO-MSC-NTFs (administered intratracheally) in a mouse model of acute respiratory distress syndrome (ARDS), the leading cause of death from coronaviruses. The LPS-induced ARDS model is an accepted model for severe human acute respiratory disease caused by coronavirus infection.

[0164] The dose is 2.0 × 10 10 The treatment was carried out by daily administration of MSCs or EXO-MSC-NTFs via an endotracheal tube at a concentration of vesicles / 1 ml (Table 2).

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

[0166] Dosage: 2.0 x 10 10EXO-MSCs or EXO-MSC-NTFs were administered daily via the endotracheal tube at a concentration of vesicles / 1 ml, starting 3 hours after LPS administration.

[0167] [Table 2]

[0168] Figure 6 shows the total severity score of acute lung injury for test groups 1 to 4. As shown in the figure, 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) for test groups 1 to 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 from test groups 1 to 4 (Figure 7D). As shown, administration of EXO-MSC-NTF significantly reduced the number of infiltrating neutrophils in the lungs after LPS administration. Furthermore, the number of neutrophils after EXO-MSC-NTF administration was not significantly different from the number of neutrophils in mice that were not administered LPS.

[0171] Figure 8 - Histopathology: Showing a multifocal distribution of perivascular infiltrates, primarily neutrophils (acute). Fibrin deposition was mild, with thickened alveolar walls in affected areas. Test Group 3 (Figure 8C) exhibited moderate to severe lung damage with an average respiratory (Resp) score of 4.4. Test Groups 1 (Figure 8A) and 2 (Figure 8B) exhibited moderate lung damage with an average respiratory (Resp) score of 3.6 and 2.5, respectively. Test Group 4 (Figure 8D) exhibited a significantly lower score with an average respiratory (Resp) score of 0.3.

[0172] 9 shows the oxygen saturation levels of test groups 1 to 4. As shown in the figure, administration of EXO-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 to 4. As shown in the figure, 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 to 4. As shown in the figure, administration of EXO-MSC-NTF significantly reduced the effect of LPS on the concentrations of IL-10 and RANTES.

[0175] 12 shows the TNF-α concentrations in the BAL fluid of test groups 1 to 4. As shown in the figure, administration of EXO-MSC-NTF significantly reduced the effect of LPS on TNF-α concentrations.

[0176] Severe COVID-19 is associated with thrombotic coagulation disorders. Its pathogenesis is related to the virus's effects on the immune system and downregulation of ACE2, which leads to elevated angiotensin II levels. Increased levels of inflammatory cytokines and angiotensin II are both known to induce tissue factor (TF) and activated neutrophils. Tissue factor (TF) may be an important mediator involved in the development of thrombotic events in COVID-19.

[0177] Another coagulation factor, thrombin-antithrombin complex (TAT), was found to be higher in non-survivors than in survivors in the early and middle stages of the disease, reflecting excessive thrombin generation. Tissue factor (TF) and TAT concentrations were examined using ELISA assays in the serum and BALF of ARDS mice treated with EXO-MSCs or EXO-MSC-NTFs.

[0178] As described above in Example 3, MSCs are induced to differentiate into MSC-NTFs (MSCs that secrete neurotrophic factors) using a medium-based approach by culturing MSCs in 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] Animals were weighed daily and were excluded from the study if their body weight decreased by 20% from baseline or by 10% or more during the study period. Additionally, animals were excluded from the study if they exhibited severe dehydration, hypoactivity, skin lesions, persistent tremors, or respiratory failure. During the study, animals had free access to food and drinking water.

[0180] To measure the content of specific proteins in MSC small extracellular vesicles (sEVs, EXO-MSCs), 1 ml of the sEV-enriched fraction was precipitated using ExoQuick-CG (SBI, USA). The EV pellet was lysed using M-PER Mammalian Protein Extraction Reagent (ThermoFischer, USA) and supplemented with 1:200 Protease Inhibitor Cocktail Set III, EDTA-free (Calbiochem). After 10 min of incubation at room temperature, the lysate was frozen and then thawed twice to ensure complete lysis. The protein concentration of the lysate was measured using a BCA kit (ThermoFischer, USA) and used for ELISA analysis at concentrations 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 and 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-MSCs and EXO-MSC-NTFs were evaluated in vitro by examining the suppression of cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to activation with phytohemagglutinin (PHA). PBMCs (5 × 10 5 ) were stimulated with 10 μg / mL of PHA and then incubated with EXO-MSCs or EXO-MSC-NTFs (2 × 10 9 The cells were cultured in medium with 1000 μg of 10 ...

[0182] Addition of sEVs, EXO-MSCs, or EXO-MSC-NTFs to activated PBMCs suppressed the secretion of IFN-γ (Figure 13A) and TNF-α (Figure 13B). Although there was no significant difference between EXO-MSCs and EXO-MSC-NTFs in their ability to suppress IFN-γ secretion, EXO-MSC-NTFs significantly suppressed the secretion of TNF-α.

[0183] To investigate the differences between EXO-MSCs and EXO-MSC-NTFs that may contribute to the superior efficacy of EXO-MSC-NTF administration, we evaluated the differences in protein cargo between EXO-MSCs and EXO-MSC-NTFs from three independent donors. ELISA measurements revealed 16-fold higher AREG and over 3-fold higher LIF in EXO-MSC-NTFs compared to EXO-MSCs (Figures 14A and 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 the differences were not significant (Figures 14C and 14D).

[0184] Table 3 summarizes the differences in the main protein cargo between EXO-MSCs and EXO-MSC-NTFs.

[0185] [Table 3]

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

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

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

[0189] This is a randomized, parallel-group, open-label, multi-site study involving up to 60 subjects who developed severe novel coronavirus pneumonia (NCP) due to COVID-19 at the screening visit. After providing informed consent and signing an informed consent form, all subjects were randomly assigned to the study and observed for a total of 28 days (1 month).

[0190] Subjects who were eligible based on the inclusion and exclusion criteria were to receive intravenous administration of MSCs (80-100 M MSCs / 4 ml), intravenous administration of EXO-MSCs (at least 1.0 × 10 10 Patients were randomized to one of three cohorts: intravenous administration of MSCs (10 EXO-MSCs / 10 ml) or a combination of MSCs and EXO-MSCs, administered on days 1, 2, 3, 4, and 5, or on days 1, 3, and 5.

[0191] After the 3-day or 5-day treatment, subjects will be followed for up to 28 days. Study safety parameters, physiological parameters, and biomarkers will be acquired.

[0192] The study consists of a 5-day dosing period followed by a follow-up period until day 28 (approximately 1 month, Figure 15). Dosing occurs in a hospital's acute care unit or intensive care unit (ICU). After each dose, subjects are evaluated daily. After the 3-day or 5-day dosing period, all subjects are followed for up to 28 days for primary efficacy and safety assessments. Thus, each subject is followed for a total of approximately 28 days (1 month) from the initial visit.

[0193] Eligible subjects who meet the inclusion / exclusion criteria will be randomly assigned to one of the six cohorts in Table 4 to receive treatment.

[0194] [Table 4]

[0195] Procedure for intravenous administration of MSCs: Procedure for intravenous administration of MSCs (80-100M MSCs / 4ml). A 5ml syringe filled with 4ml of cell suspension is used. The cell suspension is then injected from the syringe into a 100ml Plasmalyte A bag and administered intravenously to the subject over one hour.

[0196] Procedure for intravenous administration of EXO-MSCs: EXO-MSCs (at least 1.0 × 10 9 Procedure for intravenous administration of 100 exosomes (100 exosomes / 10ml). A 10ml syringe is filled with 10ml of exosomes. The exosomes are then injected from the syringe into a 100ml Plasmalyte A bag, which is then administered intravenously to the subject over one hour.

[0197] Procedure for intravenous administration of a combination of MSCs and EXO-MSCs: MSCs (80-100 M MSCs / 4 ml) and EXO-MSCs (at least 1.0 × 10 9 The procedure for intravenous administration of the combination MSC product and exosome product (100ml of exosomes / 10ml) will be as follows: The MSC product and exosome product will each be injected into separate 100ml Plasmalyte-A bags and infused into the subject over one hour, with at least two hours between each infusion.

[0198] It should be understood that cells (MSCs, MSC-NTFs) can be administered intravenously, while exosomes (EXO-MSCs, EXO-MSC-NTFs) can be administered intravenously, intratracheally, or intranasally (inhaled through the nostrils).

[0199] Subjects will be screened and eligible subjects will be enrolled. Each subject will be followed for approximately 28 days, with efficacy and safety assessments conducted over approximately 28 days or at the end of the study.

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

[0201] This dose was 100-125 × 10 in over 200 ALS and MS patients. 6 Intrathecal transplantation of MSC-NTFs was shown to be safe. EXO-MSCs are derived from the same MSC cell source.

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

[0203] Subjects who met all of the following criteria were allowed to participate in the study: 1. Men and women aged 18 to 75 years at the time of screening. 2. Individuals with laboratory-confirmed 2019-nCoV infection by reverse transcription-polymerase chain reaction (RT-PCR) from any diagnostic sampling source. 3. Subjects with baseline chest computed tomography confirmed pneumonia consistent with COVID-19. 4. Subjects who meet any one of the following criteria: (1) shortness of breath (RR 30 or more times per minute), (2) finger oxygen saturation at rest 93% or less, (3) arterial blood oxygen partial pressure (PaO2) / oxygen absorption concentration (FiO2) 300MMHG or less, or (4) lung imaging showing focus progression of more than 50% within 24 to 48 hours. 5.Persons with ARDS associated with COVID-19 infection. 6. Those who have a medical need for endotracheal intubation and mechanical ventilation. 7. The patient is receiving maximum intensive medical treatment as determined by a physician.

[0204] Alternatively, the main eligibility criteria are 1 to 5 below. 1. Men and women aged 18 to 75. 2. Individuals with laboratory-confirmed 2019-nCoV infection by reverse transcription-polymerase chain reaction (RT-PCR) from any diagnostic sampling source. 3. Subjects with acute onset of ARDS as defined by the Berlin Criteria, including the following (1) to (5): (1) Subjects with pneumonia or worsening respiratory symptoms within one week of known clinical impairment. (2) Subjects with bilateral pulmonary opacities not explained by pleural effusion, lobar / lung collapse, or nodules on chest X-ray or CT scan. (3) Subjects with pulmonary edema not adequately explained by heart failure or fluid overload. (4) Subjects with hypoxemia defined as a PaO2 / FiO2 ratio of less than 300 mmHg. 4. Subjects have radiological pulmonary changes consistent with COVID-19 ARDS (consolidation, ground-glass opacities, or bilateral pulmonary infiltrates) on a baseline high-resolution chest computed tomography (HRCT) obtained within 5 days of initiating treatment. 5. Subjects with respiratory failure defined as blood oxygen saturation (SpO2) less than 93%.

[0205] Subjects who met any of the following criteria at the time of screening evaluation were excluded from participation in the study: 1. If you have previously undergone stem cell therapy. 2. A history of malignancy within the past 5 years, excluding localized non-melanoma skin cancer (with no evidence of metastasis, significant invasion, or recurrence within 3 years of the screening test (first visit)). 3. Currently using immunosuppressive medication or have used such medication within 6 months of study enrollment, but this does not include the therapeutic use of steroids or other treatments deemed necessary for the management of COVID-19. 4. If you are a pregnant or breastfeeding woman. 5. Informed consent could not be obtained from the patient or an authorized family member.

[0206] Alternatively, the main exclusion criteria are 1 to 5 below. 1. Informed consent could not be obtained from the patient or an authorized family member. 2. Currently using chronic immunosuppressive medications or have used such medications within 6 months of study enrollment, but this does not include the therapeutic use of steroids or other treatments deemed necessary for the management of COVID-19. 3. If you are a pregnant or breastfeeding woman. 4. If you have previously undergone stem cell therapy. 5. If you are an organ transplant recipient.

[0207] Test evaluation

[0208] Bronchoalveolar lavage and blood sampling for biomarker assessment: Bronchoalveolar lavage and serum sampling for biomarker detection.

[0209] COVID viral load test: Nasopharyngeal swab to check the COVID virus genome (as per hospital protocol).

[0210] High-resolution chest CT scan: High-resolution chest computed tomography (HRCT) according to hospital protocol.

[0211] Clinical Laboratory Safety Studies: Clinical laboratory safety studies will be monitored throughout the study at Visits 1-9.

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

[0213] Serum pregnancy test: hCG

[0214] Blood chemistry: sodium (Na), potassium (K), calcium (Ca), bicarbonate (HCO3), 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 ration (INR).

[0216] Urinalysis: specific gravity, pH, glucose, protein, ketones, blood.

[0217] Vital sign measurements (blood pressure, temperature, pulse, respiratory rate, etc. after sitting for at least 3 minutes) will be monitored at screening (Visit 1) and at all visits through the final visit.

[0218] A standard 12-lead electrocardiogram will be performed at visit 1. The electrocardiogram results should be read manually, preferably by a cardiologist, and the results entered into an electronic case report form (eCRF).

[0219] Daily assessments: safety and adverse events including clinical course of ARDS, vitals, laboratory assessments (CBC with differential, platelet count, BUN, creatinine, LDH, PT, PTT, INR, ferritin, D-dimer, ALT, AST, pH, lactate, CK).

[0220] Respiratory physiological parameters (PaO2 / FiO2 ratio).

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

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

[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-γ.

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

[0225] Day 10 (in addition to daily assessments): Nasopharyngeal swab (NP) to confirm the presence of COVID viral genome. High-resolution chest computed tomography (HRCT) to assess changes in lung imaging abnormalities compared to baseline (pre-dose, Day 1). Serum / blood biomarker collection.

[0226] Day 28 (in addition to daily assessments): Safety and adverse events (proportion of subjects with treatment-related adverse events assessed by CTCAE v4.0). Number of ventilator-free days during the study. Number of ICU-free days during the study. Overall survival / mortality (proportion of deaths from all causes). Clinical Critical Treatment Index.

[0227] Pre-administration visit

[0228] Visit 1: Screening and randomization visit (Day 0).

[0229] Written informed consent (ICF) must be obtained from the subject or their legally authorized representative (LAR) before any study-specific screening assessments are performed.

[0230] The following assessments and procedures will be performed: Signed informed consent (obtained by the Principal Investigator (PI) and Sub-Investigator (Sub-I)). Collection of demographic data. Medical history. COVID history and date of diagnosis. Nasopharyngeal swab (NP) to confirm the COVID viral genome. High-resolution chest computed tomography (HRCT). Bronchoalveolar lavage (BAL) to collect biomarkers. Standard 12-lead electrocardiogram (ECG). Confirmation of past medication. Measurement of vital signs (blood pressure, temperature, pulse rate, respiratory rate, etc.). Respiratory variables (minute ventilation, respiratory rate, oxygenation index, PEEP value).

[0231] Clinical Critical Treatment Index: no activity limitations, discharged = score 1; activity limitations = score 2; hospitalized, no oxygen therapy = score 3; oxygen therapy via mask or nasal cannula = score 4; noninvasive 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 was collected for hematological tests (CBC (hematology panel): hemoglobin, hematocrit, white blood cell count (and 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 collection (baseline) for serum biomarker analysis.

[0234] Blood collection for serum pregnancy testing (female subjects of childbearing potential).

[0235] Urinalysis (specific gravity, pH, glucose, protein, ketones, blood).

[0236] Determine study eligibility and review inclusion / exclusion criteria.

[0237] Randomization of eligible subjects.

[0238] Administration visit

[0239] Visits 1, 2, 3, 4, and 5 (Days 1, 2, 3, 4, and 5) or Visits 1, 3, and 5 (Days 1, 3, and 5): To be included in the study, a subject must continue to meet all eligibility criteria and no exclusion criteria from the study start date. If a subject's clinical status changes between screening (Day 0) and Visit 1 (Day 1), some or all of the screening assessments must be repeated to assess the subject's eligibility.

[0240] Pre-dose assessment (up to 2 hours before administration) will include the following: Measurement of vital signs (systolic blood pressure (mmHg); temperature, pulse, respiratory rate (per minute); SpO2 scale 1 (%), SpO2 scale 2 (%); use of air or oxygen, etc.). Clinical and radiological progression of ARDS. Safety and adverse events, including consideration of concomitant medications. Blood will be drawn for hematology (PT, PTT, INR), coagulation tests, and biochemistry tests (sodium, potassium, chloride, glucose, BUN, creatinine). Blood / serum will be drawn to collect biomarkers (on days 1, 2, 3, 4, and 5 or approximately 6 hours after administration on days 1, 3, and 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). Cytokine-secreting immune cells: CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, CXCR3+ NK cells.

[0241] After administration, subjects underwent the following: Vital signs were monitored at 2 hours (±15 minutes), 8 hours (±15 minutes), and 20 hours (±30 minutes) after transplant; blood was drawn at 20 hours (±30 minutes) after transplant for biomarker assessment; Sequential Organ Failure Assessment (SOFA) score; Apache II (Acute Physiology and Chronic Health Evaluation II) score; Glasgow Coma Scale (GCS) score; and adverse event (AE) assessment.

[0242] Post-administration follow-up

[0243] Visit 7 (Day 10) and Visit 8 (Day 22): At the follow-up visits on Days 10 and 22, subjects underwent the following: Review of concomitant medications. Review of adverse events. Measurement of vital signs (blood pressure, temperature, pulse, respiratory rate, etc.). Nasopharyngeal swab (NP) to confirm COVID viral genome. High resolution chest CT: change 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 ECG. Review of concomitant medications. Review of adverse events (AEs).

[0244] Blood was collected for hematological tests (CBC (hematology panel): hemoglobin, hematocrit, white blood cell count (and 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).

[0245] Urinalysis (specific gravity, pH, glucose, protein, ketones, blood).

[0246] Visit 9: Day 28 (± 5 days) follow-up. At Visit 9 post-dose follow-up, all subjects underwent the following: a review of concomitant medications; a review of adverse events; and vital signs (blood pressure, temperature, pulse, respiratory rate, etc.).

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

[0248] The following were recorded: number of days on ventilator and number of subjects successfully weaned from ventilator; number of days in ICU; mortality and percentage of all-cause deaths; number of days free of organ failure (cardiovascular, coagulation, hepatic, renal) by day 28; an increase of SpO2 / FiO2 of 50 or more compared to its nadir; time to improvement of oxygenation in hospital for at least 48 hours; Clinical severity rating index (time to improvement).

[0249] No activity limitations, discharged = score 1; activity limitations = score 2; hospitalized, no oxygen therapy = score 3; oxygen therapy via mask or nasal cannula = score 4; noninvasive 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: All subjects receiving a dose or partial dose will be followed for safety and efficacy for approximately 28 days. Adverse events (AEs) and serious adverse events (SAEs) will be monitored.

[0251] Investigational Drug Information. Mesenchymal Stem Cell (MSC) Product Overview

[0252] MSCs are provided in ready-to-use dosing packages with appropriate primary and secondary labels. The dosing package consists of one 5 mL syringe for intravenous administration. Each dosing package contains 100 x 10 6 It consists of a ready-to-inject syringe containing 100 cells of allogeneic MSCs.

[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 dosage packages will be delivered to the medical center in a shipping system container designed to maintain a temperature of 2-8°C during shipment. The product (investigational drug) will be administered to patients within the set expiration period.

[0255] Alternatively, the dosing package contains 130 x 10 6 The treatment consists of one cryotube containing 100 allogeneic MSC cells per tube. The cryotube is shipped in liquid nitrogen vapor and thawed at the patient's bedside.

[0256] MSCs are administered intravenously by injecting 4 ml of cell suspension from a syringe into a bag filled with 100 ml of Plasmalyte A and infusing over 1 hour.

[0257] The patient received three fluid infusions without any significant adverse events. Laboratory tests confirmed a decrease in CRP and d-dimer levels. The patient still required oxygen inhalation, but after administration, the oxygen saturation decreased from 40 L to 30 L and the flow increased from 92% to 97%. The chest infiltrates persisted. Two PCR tests for COVID-19 virus were negative.

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

[0259] EXO-MSCs are provided in ready-to-use dosing packages with appropriate primary and secondary labels. The dosing package consists of one 10 mL syringe for intravenous administration. Each dosing package contains at least 1.0 x 10 cells in 10 mL. 9 It consists of a ready-to-inject syringe containing EXO-MSCs.

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

[0261] The dosage packages will be delivered to the medical center in a shipping system container designed to maintain a temperature of 2-8°C during shipment. The product (investigational drug) will be administered to patients within the set expiration period.

[0262] Alternatively, the administration package will consist of one cryotube containing 10 ml of MSC-exosomes. The cryotube will be shipped on dry ice.

[0263] EXO-MSC-exosomes were administered intravenously by injecting 10 ml of cell suspension from a syringe into a bag filled with 100 ml of Plasmalyte A, and the infusion was carried out over 1 hour.

[0264] Prior and concomitant treatment

[0265] Prior Therapy: Subjects who have received any prior cell therapy will be excluded from this study. To minimize the amount and impact of missing data, the investigator will make every reasonable effort to collect primary efficacy and safety data on subjects who discontinue treatment or who withdraw from the study. All medications taken before the first dose will be recorded as prior therapy.

[0266] Concomitant and Exclusionary Therapies: Concomitant medications are medications administered to subjects during or after the first dose. All concomitant medications will be recorded. Current use of immunosuppressant medications or use of such medications within 6 months of study enrollment will exclude subjects from the study. However, this does not include use of therapeutic agents such as corticosteroids that may be necessary for the management of COVID-19.

[0267] Safety Reporting: The study will collect adverse events (AEs) and serious adverse events (SAEs) from the time of informed consent until the end of the study (Visit 9 or early discontinuation visit). Outcomes. The following terms will be used in this study: fatal; not recovered / not resolved; recovering / resolving; recovered / resolved; recovered / resolved with sequelae; unknown; clinically significant laboratory abnormalities.

[0268] Laboratory abnormalities determined by the investigator to be clinically significant will be reported on the AE eCRF. A clinically significant abnormality is one identified as having changed significantly from the screening visit and that, in the investigator's judgment, requires a change in management. This change may include further monitoring of the laboratory test, initiation of other diagnostic tests or procedures, a change in ongoing medication, or the administration of a new medication. Whenever possible, the etiology of the abnormal finding (e.g., anemia) will be recorded on the eCRF. If clinically indicated, repeat additional testing and / or other evaluations as necessary to determine the significance and etiology of the abnormal result.

[0269] Study Discontinuation: Study or Study Site Discontinuation

[0270] Circumstances may arise during a clinical trial that may prompt the termination of the trial or the withdrawal of a site's participation. These circumstances may include, but are not limited to: The discovery of an unexpected, serious, or unacceptable risk to a subject enrolled in the trial The Data Safety Monitoring Board (DSMB) decides to recommend suspending or terminating the trial The sponsor decides to suspend, terminate, or shorten the trial

[0271] A clinical trial may be terminated at a site if: The investigator fails to enroll eligible subjects in the trial; The investigator fails to follow the International Council for Harmonisation (ICH)-Good Clinical Practice (GCP) guidelines or FDA guidelines and regulations; The site submits false information to the sponsor, clinical monitor, FDA, or IRB; The site fails to comply with protocol requirements; The investigator, his / her institution, or other parties at the site have a conflict of interest that adversely affects the integrity of the trial; The institution or IRB is subject to a valid regulatory investigation.

[0272] Subject Withdrawal from the Study: Subjects may withdraw from the study for any reason at any time during the study period without repercussion. The investigator will document the reason / circumstances for withdrawal in a timely manner (preferably within 24-48 hours) in the appropriate eCRF.

[0273] A subject may discontinue the trial for any of the following reasons: For any reason related to safety or tolerability At the subject's request For any reason as determined appropriate by the investigator For any reason as determined appropriate by the sponsor

[0274] Subjects who discontinue the study for any reason will be followed up for all relevant evaluations of safety and efficacy, including the collection of clinical and laboratory evaluations as specified in this protocol.

[0275] The investigational site must record the reason on the study termination page of the eCRF in the electronic database. The record must include the date the subject withdrew consent (withdrawn from the study) and the reason for withdrawal. The recorded date will be considered the date of last contact and therefore the subject's last day of study. If the investigational site is aware of any adverse events or serious adverse events (SAEs) that occurred within 12 weeks of the last dose despite the study being discontinued, they should record this in the adverse event log in the database.

[0276] Temporary Suspension of Clinical Trial: A clinical trial may be temporarily suspended if a serious adverse event (SAE), significant intercurrent illness, or scheduling issues with cell manufacturing or patient visits occur.

[0277] Clinical endpoints

[0278] Primary endpoint: Safety; The primary endpoint is to evaluate the safety and tolerability of allogeneic MSCs and / or EXO-MSCs administered intravenously for 5 consecutive days or 3 consecutive days every other day. Safety and adverse events (proportion of subjects with study-related adverse events as assessed by CTCAE v4.0).

[0279] Secondary endpoints: Changes in BAL and blood biomarkers: The efficacy of MSC and EXO-MSC cells will be assessed by changes in BAL and blood biomarkers after administration. BAL and blood samples will be collected according to an assessment schedule, and biomarkers will be assessed before each administration throughout the trial to assess their association with MSC and EXO-MSC administration. Time to improvement in Clinical Severity Rating Index. Time to improvement in oxygenation for at least 48 hours in the hospital. Length of stay in ICU. Mortality, proportion of all-cause deaths. Number of days on mechanical ventilation and number of subjects successfully weaned from mechanical ventilation.

[0280] Statistical methods and sample size determination

[0281] Sample Size Determination: No formal sample size calculation will be performed. Efficacy and safety data from 60 subjects will provide information to help design future randomized clinical trials.

[0282] Statistical Methods: Summary of continuous variables includes sample size, mean, standard deviation, median, minimum, and maximum. Minimum and maximum values ​​are reported with the same precision as the raw values; mean, standard deviation, and median are presented with one additional decimal point beyond that reported for the raw values. Summary of discrete variables includes frequencies and percentages. All percentages are presented rounded to two decimal points (i.e., XX.X%). The baseline visit is defined as the last non-missing measurement before the start of study drug administration (first dose at Visit 1, Day 0).

[0283] A detailed statistical analysis plan (SAP) will be completed before the first subject receives the dose.

[0284] Analysis population

[0285] The primary, secondary, and exploratory efficacy endpoints will be analyzed using the modified intent to treat (mITT) population and the efficacy evaluable (EE) population. In this study, the mITT population is defined as all subjects who received at least one dose and had at least one post-assessment baseline. Baseline is defined as the most recent assessment before receiving the first dose at Visit 2 (Day 1). The EE population is defined as a subset of the mITT population who received all five doses and did not have any critical protocol deviations that would affect efficacy assessments. If the EE population is the same as or very similar to the mITT population, analyses will be performed only on the mITT population.

[0286] All safety analyses were performed on the safety population, defined as all subjects who were enrolled and received at least one dose.

[0287] Efficacy Analyses. Efficacy analyses were performed on the mITT and EE populations as described above.

[0288] Safety Analyses. All safety analyses were based on the safety population.

[0289] All adverse events (AEs) were coded into system organ classes (SOCs) and preferred terms (PTs) using the Medical Dictionary of Clinical Terms (MedDRA®). The number of treatment-emergent adverse events (TEAEs) and the number of subjects (with percentages) who experienced TEAEs were tabulated by SOC and PT.

[0290] A TEAE is an AE that occurs for the first time after initiation of treatment, or, if it occurred before treatment, worsens in severity after initiation of treatment.

[0291] Categories of TEAEs, TEAEs by severity, administration-related TEAEs, and serious TEAEs are summarized separately.

[0292] When assessing changes in safety parameters, baseline is defined as the last measurement taken before the start of the first dose.

[0293] Abnormalities in hematology, blood chemistry, and electrocardiogram evaluations are summarized.

[0294] HRCT will be assessed for study safety at baseline and at the end of the study.

[0295] Biomarker Analysis: Bronchoalveolar lavage fluid (BAL) and / or blood samples will be analyzed to determine the relationship between biomarker concentrations and clinical outcomes at each visit. Additionally, the relationship between biomarkers and clinical outcomes will be evaluated to determine whether biomarkers can predict trial outcomes. Please see the SAP for details of the analysis.

[0296] Example 7: Clinical 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 cases per 100,000 population), with an acute mortality rate of 30-70%. Potential benefits of EXO-MSC-NTF therapy include reduced mortality, shorter ICU or hospital stays, improved ventilation status, and reduced need for ventilatory support. ARDS is associated with shock, sepsis, pneumonia (including COVID-19), blood transfusions, gastric aspiration, and trauma.

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

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

[0302] Chronic obstructive pulmonary disease (COPD) affects 15 million people in the United States annually (44.3 cases per 100,000 population). In severe cases of 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, shorter ICU or hospital stays, improved ventilation status, and reduced need for ventilatory support.

[0303] Other animal models

[0304] Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in extremely preterm infants. BPD disrupts lung development and causes severe long-term respiratory complications that extend beyond childhood and into adulthood. Potential for understanding BPD and developing therapeutic strategies has been gained from large (baboon, sheep, pig) and small (rabbit, rat, mouse) animal models. These models primarily aim to induce alveolar simplification similar to that seen in infants with BPD.

[0305] Various mouse models of BPD primarily focus on hyperoxia-induced lung injury, although hypoxic, hypoxic / hyperoxia-induced, inflammation-induced, and transgenic models also exist.

[0306] Animal models of chronic obstructive pulmonary disease (COPD) are primarily induced in mice, guinea pigs, and rats. In most studies, these models are induced by exposure to cigarette smoke (CS), intratracheal lipopolysaccharide (LPS), or intranasal elastase. Studies vary in the time course and dose of inducer used. The primary parameters measured in most studies are lung pathology and lung inflammation (both inflammatory cells and inflammatory mediators), and in the few published studies, tracheal reactivity (TR) (Ghorani V, Boskabady MH, Khazdair MR, Kianmeher M. Experimental animal models for COPD: a methodological review. Tob Induc Dis. 2017 May 2;15:25; incorporated herein by reference).

[0307] EXO-MSC-NTFs exert their 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 alveolar type II epithelial cells. AREG regulates lung recovery and fibroblast function in mice after exposure to agricultural organic dust, presumably 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 lung during viral pneumonia. LIF is also reduced by chronic smoking.

[0309] Test objectives

[0310] The purpose of this study was to examine the efficacy of bone marrow-derived mesenchymal stem cells (MSCs) and EXO-MSC-NTFs (intratracheally or by aerosol inhalation) in the bleomycin mouse model, another mouse model of inflammation and fibrosis.

[0311] Bleomycin, a chemotherapy antibiotic produced by the bacterium Streptomyces verticillus, is used to induce experimental pulmonary fibrosis. Primarily after intratracheal administration, it induces inflammatory and fibrotic responses within a short period of time. Initially, the concentration of proinflammatory cytokines increases, followed by increased expression of profibrotic markers and collagen accumulation, which peak around day 14.

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

[0313] Compared to the control (PBS), MSCs are expected to improve all clinical parameters tested, and EXO-MSC-NTFs are expected to enhance the effect.

[0314] Study design

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

[0316] C57bl mice were given a single intratracheal injection of 3 U / kg of bleomycin sulfate solution to induce lung injury.

[0317] 2. Administration

[0318] Exosomes were administered intratracheally during the inflammatory phase (days 1 and 5) or the fibrotic phase (days 7 and 10), and the effects of exosomes on inflammation and fibrosis were evaluated separately.

[0319] Additionally, one test group of mice was administered by inhalation for initial evaluation of this route of administration (RoA).

[0320] 3. Test group

[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 pulmonary fibrosis score 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). Fibrosis and cytokine mRNA panel expression in lung tissue (NanoString analysis). ·Collagen content of lung tissue.

[0324] result

[0325] As a result, favorable effects on oxygen saturation and body weight were confirmed in mice receiving intratracheal administration of EXO-MSCs and EXO-MSC-NTFs compared to the control group.

[0326] Compared to the control, a significant improvement in oxygen saturation was provided in mice administered EXO-MSC-NTF by both administration schedules (days 1 and 5 (FIG. 16A), days 7 and 10 (FIG. 16B)), with EXO-MSC-NTF demonstrating superior efficacy compared to EXO-MSC on the day 1 and day 5 administration schedules. EXO-MSC-NTF administration by inhalation provided a significant oxygenation benefit compared to the control (FIGS. 16C and 16F).

[0327] Only the administration schedule of EXO-MSC-NTFs administered on days 1 and 5 resulted in a significant improvement in weight gain (Figure 16D).

[0328] The above description of the specific embodiments should make the general nature of the present invention sufficiently clear. Thus, others, applying their current knowledge, can easily modify and / or adapt the above specific embodiments for various applications without undue experimentation and without departing from the general concept. Therefore, such modifications and adaptations should be understood and are intended to be within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. 1. A pharmaceutical composition for treating a pulmonary disease or symptom thereof in a patient in need thereof, comprising: (a) Multiple neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs); (b) EXO-MSC-NTFs, defined as multiple small extracellular vesicles (sEVs) derived from neurotrophic factor-secreting mesenchymal stem cells (MSC-NTFs), and (c) a combination of neurotrophic factor-secreting mesenchymal stem cells (MSC-NTF) and EXO-MSC-NTF; and an active agent selected from the group consisting of: The NTFs include vascular endothelial growth factor (VEGF), amphiregulin (AREG), and leukemia inhibitory factor (LIF); The pharmaceutical composition is administered to said patient using a therapeutically effective regimen.

2. 2. The pharmaceutical composition of claim 1, The pulmonary disease comprises a viral pulmonary infection or a non-viral pulmonary infection.

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

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

5. 2. The pharmaceutical composition of claim 1, The pharmaceutical composition comprises about 10 ９ ~about 10 １３ A pharmaceutical composition comprising EXO-MSC-NTF.

6. The pharmaceutical composition according to claims 1 to 5, The pharmaceutical composition comprises a combination of MSC-NTF and EXO-MSC-NTF.

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

8. 8. The pharmaceutical composition of claim 7, The repeated administration comprises administration on at least five different days.

9. 8. The pharmaceutical composition of claim 7, The repeated administrations include administrations on days 1, 3, and 5.

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

11. 11. The pharmaceutical composition of claim 10, The pharmaceutical composition, wherein the excipient comprises Plasmalyte A, DMEM, CryoStor® CS10 freezing medium, or any combination thereof.

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

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

14. The pharmaceutical composition according to any one of claims 1 to 13, The symptom is 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, oxygen desaturation, muscle pain, joint pain, sore throat, headache, chills, nausea, vomiting, nasal congestion, diarrhea, inflammation, and fibrosis.

15. 15. The pharmaceutical composition of claim 14, The condition is pneumonia, acute respiratory distress syndrome (ARDS), or a combination thereof.

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

17. 5. The pharmaceutical composition of claim 4, The EXO-MSC-NTFs exhibited the following characteristics compared to their corresponding EXO-MSCs: (i) is substantially reduced in 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) A pharmaceutical composition substantially enriched in at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

18. 18. The pharmaceutical composition of claim 17, The EXO-MSC-NTFs exhibited the following characteristics compared to their corresponding EXO-MSCs: (i) is substantially reduced in 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) A pharmaceutical composition substantially enriched in at least one protein selected from the group consisting of P02748, P08476, P08254, P05067, P15514, P07602, P20809, CON_P13645, P13645, and P01857.

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

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

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