Treatment of virus-induced acute respiratory distress syndrome

Abstract

Providing treatment for virus-induced acute respiratory distress syndrome [Solution] The present invention relates to a method for treating virus-induced acute respiratory distress syndrome (ARDS) in a subject having ARDS by administering multipotent adult progenitor cells (MAPCs), wherein the MAPCs are non-embryonic stem, non-germinal cells with broad differentiation potential and long-term replication ability. The virus that induces ARDS may be a betacoronavirus (e.g., severe acute respiratory syndrome (SARS) coronavirus or Middle East respiratory syndrome (MERS) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).

Description

[Background technology]

[0001] background As more information emerges about the current COVID-19 pandemic and its etiology, it is clear that an exacerbated and uncontrolled immune response plays a detrimental role in the disease pathology. COVID-19 can cause severe pneumonia leading to ARDS. It has been suggested that patients with ARDS caused by severe COVID-19 may develop cytokine storm syndrome (Mehta et al., Lancet 2020, 395(10229):1033-1034). In this case, cytokines induced by T cells and macrophages (e.g., IL-6, TNFα, IFNγ, IL-2, IL-7, and IL-17) have been reported to be elevated in the plasma of patients with severe COVID-19 compared to mild COVID-19 cases (Huang et al., Lancet 2020, 395(10223):497-506; Qin et al., Clin. Infect. Dis. 2020, 71(15):762-768; Chen et al., Lancet 2020, 395(10223):507-513). Furthermore, chemokines CXCL10, CCL2, and CCL3, which are involved in the recruitment of T cells and monocytes / macrophages, are also elevated (Mehta et al., Lancet 2020, 395(10229):1033-1034; Huang et al., Lancet 2020, 395(10223):497-506; Qin et al., Clin. Infect. Dis. 2020, 71(15):762-768). Enhancement gene expression of some of these cytokines has also been observed in bronchoalveolar lavage fluid from COVID-19 patients. This suggests an increased inflammatory response in the lungs (Xiong et al., Emerg. Microbes Infect. 2020, 9(1):761-770; Liao et al., medRxiv 2020, Nature Medicine 2020, 26:842-844).

[0002] Analysis of the frequency of immune cells in peripheral blood showed that patients with severe COVID-19 suffered from lymphopenia of both CD4 and CD8 T cells. These patients' T cells expressed high levels of T cell exhaustion markers (e.g., PD-1 and Tim-3), along with increased expression of activation markers (e.g., CD69 and CD38) (Zhou et al., bioRxiv 2020, doi: https: / / doi.org / 10.1101 / 2020.02.12.945576). RNA-seq analysis of COVID-19 PBMCs showed an enlarged apoptotic signaling pathway, suggesting that increased lymphocyte apoptosis may be the cause of lymphopenia (Xiong et al., Emerg. Microbes Infect. 2020, 9(1):761-770). T cell exhaustion arises as a response to chronic antigenic stimulation, and it is widely described among chronic viral infections. It has been proposed that during COVID-19, T cells become exhausted due to the increased and sustained pro-inflammatory response, leading to T cell apoptosis. Bronchoalveolar CD8 T cells were observed to be reduced in patients with severe COVID-19, along with limited clonal expansion, compared to patients with mild disease. This suggests impaired CD8 T cell response. Excessive lymphopenia in these patients also resulted in a reduction of regulatory T cells, which contributed to the observed, uncontrolled inflammatory response (Qin et al., Clin. Infect. Dis. 2020, 71(15):762-768). While serum lymphocytes are reduced in severely ill patients, monocytes / macrophages appear to be highly enriched in the lungs of severely ill COVID-19 patients (Liao et al., medRxiv 2020, Nature Medicine 2020, 26:842-844). Phenotypic analysis of peripheral blood monocytes from these patients showed that these cells were enlarged, highly activated, and secreted higher levels of pro-inflammatory cytokines than in mild COVID-19 and healthy controls (Zhang et al., J. Leukoc. Biol. 2021, 109(1):13-22, MedRxiv 2020). Intermediate (CD14 +and CD16 + ) and non-classical (CD14 - CD16 + An increase in monocytes was observed in ICU patients. This suggests that the monocyte composition is altered in patients with severe COVID-19 (Liao et al., medRxiv 2020, Nature Medicine 2020, 26:842-844). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Mehta et al., Lancet 2020, 395(10229):1033-1034 [Non-Patent Document 2] Huang et al., Lancet 2020, 395(10223):497-506 [Non-Patent Document 3] Qin et al., Clin. Infect. Dis. 2020, 71(15):762-768 [Non-Patent Document 4] Chen et al., Lancet 2020, 395(10223):507-513 [Non-Patent Document 5] Xiong et al., Emerg. Microbes Infect. 2020, 9(1):761-770 [Non-Patent Document 6] Liao et al., medRxiv 2020, Nature Medicine 2020, 26:842-844 [Non-Patent Document 7] Zhou et al., bioRxiv 2020, doi: https: / / doi.org / 10.1101 / 2020.02.12.945576 [Non-Patent Document 8] Zhang et al., J. Leukoc. Biol. 2021, 109(1):13-22, MedRxiv 2020 [Overview of the Initiative] [Means for solving the problem]

[0004] Summary of the Invention Patients with severe COVID-19 have a dysregulated hyperinflammatory response characterized by increased T cell and monocyte / macrophage activation, which is evident to contribute to and may be the cause of the disease pathology. The inventors investigated whether multipotent adult progenitor cells (MAPCs) have the ability to modulate the unregulated immune response, thereby re-establishing immune homeostasis and promoting tissue repair in virus-induced acute respiratory distress syndrome (ARDS), specifically in COVID-19-induced ARDS. The inventors used a commercially available preparation called "MultiStem®," based on MAPCs, disclosed by Jiang et al., Nature 2002, 418:41-9. Evidence exists that MultiStem® cells reduce dysregulated, prolonged T cell activation and proliferation (Reading et al., J. Immunol. 2013, 190:4542-4552; Reading et al., Molecular Therapy 2015, 23(11):1783-1793; Yang et al., Stem Cells 2017, 35:1290-1302; Carty et al., Front. Immunol. 2018, 9:645 (doi: 10.3389 / fimmu.2018.00645). Based on this, the inventors considered the possibility of preventing T cell exhaustion by administering MultiStem® to patients with moderate to severe ARDS (Walker et al., J. Neuroinflammation 2012, 9:228; Reading et al., J. Immunol. 2013, 190(9):4542-4552; Kovacsovics-Bankowski et al., Cell Immunol. 2009, 255(1-2):55-60). Furthermore, there are reports that MultiStem® cells promote the differentiation of regulatory T cells (Walker et al., J. Neuroinflammation 2012, 9:228; Reading et al., J. Immunol. 2013, 190(9):4542-4552; Reading et al., Mol. Ther. 2015, 23(11):1783-1793; Yang et al., Stem Cells 2017, 35(5):1290-1302). Therefore, the inventors believe that administration of MultiStem® can alleviate ARDS and drive the proliferation of AT2 cells (Mock et al., Mucosal Immunol. 2014, 7(6):1440-1451). Furthermore, it has been reported that MultiStem® cells reduce the monocyte / macrophage pro-inflammatory profile by decreasing the production of pro-inflammatory cytokines and the expression of pro-inflammatory markers (Walker et al., J. Neuroinflammation 2012, 9:228; DePaul et al., Sci. Rep. 2015, 5:16795; Busch et al., J. Neurosci. 2011, 31(3): 944-953), induce differentiation of M2 anti-inflammatory monocyte / macrophage, thereby promoting lung tissue repair by removing pathogens and cellular debris while limiting the inflammatory response (Walker et al., J. Neuroinflammation 2012, 9:228; DePaul et al., Sci. Rep. 2015, 5:16795; Busch et al., J. Neurosci. 2011, 31(3): 944-953). Therefore, the inventors investigated whether the MultiStem® immunomodulatory properties could help patients with moderate to severe COVID-19 reduce their dysregulated immune response by dissipating inflammation, restoring homeostasis, and promoting tissue repair. [Modes for carrying out the invention]

[0005] Detailed explanation In one embodiment, a method for treating virus-induced ARDS comprises the step of administering MAPC to a subject having virus-induced ARDS in a sufficient amount and for a sufficient amount of time via an effective route to treat the ARDS.

[0006] In one embodiment, the virus that induced the above-mentioned ARDS is Betacoronavirus.

[0007] In one embodiment, the virus that induced the above-mentioned ARDS is severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) coronavirus, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0008] In one embodiment, the subject is a human being.

[0009] In one embodiment, the parameters measured in the subject from the first administration are as follows: mortality on days 28 and 60; number of days off the ventilator by day 28; survival and days off the ventilator by day 28, combining days of death and days without invasive mechanical ventilation; percentage of subjects surviving and not using a ventilator on days 7 and 28; change in the Sequential Organ Failure Assessment score from day 0 (before injection) to days 3 and 7; number of days off the ventilator from day 0 to day 60; number of days out of the ICU from day 0 to days 28 and 60; white blood cell populations on days 0, 3, and 7; inflammatory biomarkers on days 0, 1, 3, and 7; and changes in oxygenation levels (PaO2 / FiO2 ratio), oxygenation index, peak pressure and plateau pressure, and PEEP requirements from baseline (day 0) to days 1, 2, 3, and 7 (and days 4, 5, and 6 for subjects receiving two doses of MAPC). These can be compared to an untreated population (i.e., without MAPC injection) based on known historical averages.

[0010] In one embodiment, in the dosing regimen, 900 million to 1.2 billion MAPC cells are provided on day 0, or optionally on day 0, and an additional dose of 900 million to 1.2 billion MAPC cells is provided 72 to 96 hours after the first dose.

[0011] In one embodiment, the route of administration is intravenous.

[0012] In one embodiment, the MAPC is allogeneic.

[0013] In one embodiment, the MAPC is derived from bone marrow.

[0014] In one embodiment, the MAPC is human.

[0015] 1. Treatable viruses - influenza, coronaviruses (including SARS-CoV, MERS-CoV, SARS-CoV2), herpes simplex virus, cytomegalovirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, adenovirus, and severe systemic diseases (viral hemorrhagic fevers) that can cause ARDS complicated by direct endothelial damage or indirectly through cytokine storms / sepsis syndrome (e.g., filoviruses (e.g., Ebola, Marburg), arenaviruses (LCMV, Lassa, Fini, Lujo, etc.), bunyaviruses (Rift Valley fever virus and Crimean-Congo hemorrhagic fever virus, and hantaviruses), and flaviviruses (yellow fever virus, dengue fever virus, Japanese encephalitis virus, West Nile virus, Zika virus, etc.)).

[0016] 2. Acute respiratory distress syndrome (ARDS) - a severe form of inflammatory lung injury characterized by increased vascular permeability in the lungs. Clinically, ARDS is defined by severe hypoxemia and the presence of bilateral shadows on chest imaging that cannot be adequately explained by heart failure or fluid overload. Specifically, it is defined by the Berlin definition (or the Kigali amendment of the Berlin criteria (see, for example, Riviello et al., Am. J. Respir. Crit. Care Med. 2016, 193(1):52-59)).

[0017] 3. Berlin (and Kigali amendment) definition of ARDS - a. Timing - onset must be within 1 week of a known clinical insult or new or worsening respiratory symptoms. b. Imaging (chest X-ray or computed tomography scan or ultrasound (Kigali amendment)) - bilateral shadows consistent with pulmonary edema on chest X-ray. ​​c. Cause of edema - Respiratory failure not adequately explained by heart failure or fluid overload, as determined by the treating physician using all available data d. Oxygenation i. Mild ARDS - 200 mmHg < PaO2 / FIO2 ≤ 300 mmHg and PEEP or CPAP ≥ 5 cm H2O; or SpO2 / FIO2 ≤ 315 (Kyari Amendment) ii. Moderate ARDS - 100 mmHg < PaO2 / FIO2 ≤ 200 mmHg and PEEP ≥ 5 cm H2O iii. Severe ARDS - PaO2 / FIO2 ≤ 100 mmHg and PEEP ≥ 5 cm H2O 1. Abbreviations: CPAP, continuous positive airway pressure; FIO2, inspiratory oxygen concentration; PaO2, arterial oxygen partial pressure; PEEP, positive end-expiratory pressure.

[0018] 4. In one embodiment, the patient may be selected as follows: Dysregulated immune response - In cases of ARDS, maladaptive changes in the molecular control of an immune system process that results in damage to the alveolar-capillary barrier and causes lung injury characterized by hypoxemia, inflammation, and non-cardiogenic pulmonary edema.

[0019] 5. In one embodiment, the patient may be selected as follows: Worsening ARDS - PEEP ≥ 5 cm H2O and PaO2 / FiO2 ≤ 300 mmHg; and PaO2 / FiO2 remains < 200 mmHg or PaO2 / FiO2 does not increase beyond 100 mmHg to the PaO2 / FiO2 measured within 6 hours of the first dose of IP administration from the screening assessment or to the PaO2 / FiO2 measured within 6 hours of the second dose of IP administration from the baseline on day 0.

[0020] In other embodiments, other parameters may be addressed and / or measured. The applicant has various endpoints on which MAPC may have beneficial effects. Any of these endpoints may be measured before and / or after treatment with MAPC. Thus, MAPC may have beneficial effects against uncontrolled immune responses that play a detrimental role in disease pathology. The following are various endpoints: reducing pneumonia; reducing cytokine storm syndrome; reducing T cell and macrophage-driven cytokines (e.g., IL-6, TNF-α, IFNγ, IL-2, and IL-7); reducing CXCL10, CCL2, and CCL3 (cytokines involved in T cell and monocyte / macrophage recruitment); reducing monocyte recruitment and / or activation; reducing the inflammatory response in the lungs; reducing lymphopenia of CD4 and / or CD8 T cells; reducing T cell exhaustion markers (e.g., PD-1 and TIM-3), resulting in reduced T cell exhaustion itself; reducing the expression of activation markers (e.g., CD69 and CD38); reducing T cell apoptosis; It has an effect on apoptotic signaling pathways; in COVID patients, it affects monocytes that are enlarged, highly activated, and secrete levels of pro-inflammatory cytokines; it reduces intermediate (CD14- and CD16+) and non-classical (CD14- and CD16+) monocytes, increases regulatory T cells, and reduces monocyte activation and the secretion of pro-inflammatory cytokines from those monocytes. Therefore, any of these endpoints may be measured before and / or after MAPC treatment. If measured after treatment, MAPC may affect these parameters as described, compared to historical averages in patients without presenting ARDS, and in patients who possibly have ARDS but whose ARDS are not virus-induced.

[0021] Examples of cells include, but are not limited to, those derived from non-embryonic tissue that are neither embryonic stem cells nor germ cells, but possess some characteristics of embryonic stem cells, and that provide the effects described herein. Such cells may achieve these effects naturally (i.e., without genetic or pharmaceutically modification). However, the natural expression may be genetically or pharmaceutically modified to increase potency. In one embodiment, the stem cells may be allogeneic cells that are not HLA-matched.

[0022] The above cells may express pluripotency markers (e.g., oct4). They may also express markers associated with long-term replication ability (e.g., telomerase). Other features of pluripotency may include the ability to differentiate into more than one cell type of germ layer (e.g., two or three of the embryonic germ layers of the ectoderm, endoderm, and mesoderm). The above cells may be highly enlarged without being transformed or tumorigenic, and may even maintain a normal karyotype. In one embodiment, the above non-embryonic stem non-germinoid cells may undergo a desired number of cell doublings in culture. For example, the non-embryonic stem non-germinoid cells may undergo at least 10–40 cell doublings in culture (e.g., 30–35 cell doublings), where the above cells are untransformed and have a normal karyotype. The above cells may differentiate into at least one cell type from each of two of the embryonic lineages of the endoderm, ectoderm, and mesoderm, and may include differentiation into all three types. Furthermore, the cells described above do not have to be tumorigenic (e.g., they do not produce teratomas). If the cells are transformed or tumorigenic and it is desirable to use them for injection, such cells can be rendered in vivo incapable of tumor formation by treatments that prevent cell proliferation into tumors. Such treatments are well known in the field.

[0023] The following are examples of cellular embodiments, but are not limited to them: 1. Isolated and enlarged non-embryonic stem non-germinoid cells, wherein the cells have undergone at least 10-40 cell duplication in culture, and the cells express oct4, are untransformed, and have a normal karyotype.

[0024] 2. Non-embryonic stem non-germinoid cells of item 1, further expressing one or more of telomerase, rex-1, or sox-2.

[0025] 3. Non-embryonic stem non-germinic cells of 1 above that can differentiate into at least one cell type from at least two of the embryonic lineages of endoderm, ectoderm, and mesoderm.

[0026] 4. Non-embryonic stem, non-germinal cells of the above 3, further expressing one or more of telomerase, rox-1, or sox-2.

[0027] 5. Non-embryonic stem non-germinic cells of item 3 above that can differentiate into at least one cell type from each of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0028] 6. Non-embryonic stem non-germinoid cells of item 5 above, further expressing one or more of telomerase, rex-1, or sox-2.

[0029] 7. Isolated and enlarged non-embryonic stem non-germinoid cells obtained by culturing non-embryonic non-germinoid tissue, wherein the cells have undergone at least 40 cell duplication in culture, and the cells are untransformed and have a normal karyotype.

[0030] 8. Non-embryonic stem non-germinoid cells of item 7 above, expressing one or more of oct4, telomerase, rex-1, or sox-2.

[0031] 9. Non-embryonic stem non-germinic cells of item 7 above that can differentiate into at least one cell type from at least two of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0032] 10. Non-embryonic stem non-germinoid cells of item 9 above that express one or more of oct4, telomerase, rex-1, or sox-2.

[0033] 11. Non-embryonic stem non-germinic cells of item 9 that can differentiate into at least one cell type from each of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0034] 12. Non-embryonic stem non-germinoid cells of the above 11, expressing one or more of oct4, telomerase, rex-1, or sox-2.

[0035] 13. Isolated and enlarged non-embryonic stem non-germinoid cells, the cells having undergone at least 10-40 cell duplication in culture, wherein the cells express telomerase, are untransformed, and have a normal karyotype.

[0036] 14. Non-embryonic stem non-germinoid cells of the above 13, further expressing one or more of oct4, rex-1, or sox-2.

[0037] 15. Non-embryonic stem non-germinic cells of the above 13 that can differentiate into at least one cell type from at least two of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0038] 16. Non-embryonic stem non-germinoid cells of the above 15, further expressing one or more of oct4, rex-1, or sox-2.

[0039] 17. Non-embryonic stem non-germinic cells of 15 that can differentiate into at least one cell type from each of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0040] 18. Non-embryonic stem non-germinoid cells of the above 17, further expressing one or more of oct4, rex-1, or sox-2.

[0041] 19. Isolated, enlarged non-embryonic stem non-germinic cells capable of differentiating into at least one cell type from at least two of the embryonic lineages of the endoderm, ectoderm, and mesoderm, wherein the cells undergo at least 10-40 cell duplication in culture.

[0042] 20. Non-embryonic stem non-germinoid cells of the above 19, expressing one or more of oct4, telomerase, rex-1, or sox-2.

[0043] 21. The non-embryonic stem non-germinic cells of 19 that can differentiate into at least one cell type from each of the embryonic lineages of the endoderm, ectoderm, and mesoderm.

[0044] 22. Non-embryonic stem non-germinoid cells of the above 21, expressing one or more of oct4, telomerase, rex-1, or sox-2.

[0045] The cells described above lack the expression of HLA-DR, CD45, glyA, and CD34. These cells may express one or more of the following: CD90, CD49c, CD13, CD10, and CD29.

[0046] The cells described above may originate from bone marrow (e.g., human bone marrow).

[0047] Since stem cells can provide the effects described herein through the molecules they secrete, the various embodiments described herein for the administration of stem cells can be carried out by administering one or more of the secreted molecules (e.g., which may be present in a conditioned culture medium). In one embodiment, a conditioned medium is used instead of stem cells. Components of the medium can be separated from the culture medium components, for example, to exclude animal serum.

[0048] The stem cells described above can be prepared by isolation and culture conditions as described herein. In specific embodiments, they are prepared by culture conditions as described herein, including lower oxygen concentrations in combination with higher serum concentrations.

[0049] It should be understood that the present invention is not limited to, and is therefore subject to change, the specific methodologies, protocols, and reagents described herein. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention of this disclosure, which is defined solely by the claims.

[0050] Section headings are used in this specification solely for structural purposes and should not be construed in any way as limiting the subject matter described herein.

[0051] The methods and techniques of this application are generally carried out in accordance with the prior art, as described in the various general and more specific references that are well known in the art and that are cited and discussed throughout this specification, unless otherwise indicated. For example, Sambrook et al. Molecular Cloning: A Laboratory Manual, See also the 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0052] definition "A" or "an" means one or more; at least one. Where the plural is used herein, it generally also includes the singular.

[0053] A “cell bank” is an industry term relating to cells that are grown and stored for future use. Cells may be stored in aliquots. They may be used directly from storage or expanded after storage. This is convenient because there is a “stock” of cells that can be used for administration at any time. Since the cells may already be stored in pharmaceutically acceptable excipients, they may be administered directly or mixed with appropriate excipients when they are released from storage. Cells may be stored in a form that preserves viability, either by freezing or by other means. In one embodiment of the present invention, a cell bank is prepared in which the cells are selected for their enhanced potency to achieve the effects described herein. It may be preferable to re-assay the cells for potency after release from storage and before administration. This may be done directly or indirectly using any of the assays described herein or by other methods known in the art. Cells with the desired potency may then be administered. The bank may be prepared using autologous cells (derived from organ donors or recipients). Alternatively, the bank may contain cells intended for allogeneic applications.

[0054] In relation to the present invention, “co-administering” means administering two or more drugs together. In the context of the present invention, in one embodiment, the stem cells are administered in combination with other treatment modalities (e.g., convalescent plasma; antiviral agents including remdesivir, favipiravir, and combinations of kaletra, lopinavir and ritonavir; high doses of vitamin C; or IL-6 inhibitors including tocilizumab, siltuximab or sarilumab).

[0055] "Comprising" necessarily means including the reference without any restriction or exclusion of what else may be included, but without any other limitations. For example, "a composition containing x and y" encompasses any composition containing x and y, even if other components may be present in the composition. Similarly, "a method encompassing the step of x" encompasses any method in which x is performed, whether x is the only step in the method, just one of the steps, even if there are many other steps, and even if x is simple or complex in comparison to them. Similar phrases using "comprised of" and the root word "comprise" are used herein as synonyms for "comprising" and have the same meaning.

[0056] "To be composed of" is a synonym for "to include" or "to encompass" (see above).

[0057] "Effective dose" generally means the amount that achieves the specific desired effect described in this application. For example, an effective dose is an amount sufficient to produce a beneficial or desired clinical outcome. In the context of the present invention, generally, the desired effect is a clinical improvement that compensates for an ineffective or pathological function in the subject. The effective dose may be provided as a single dose or in divided doses that provide an effective dose in several doses. The precise determination of what is considered an effective dose may be based on individual factors for each subject, including the severity of the disease / defect, the patient's health status, age, etc. A person skilled in the art may determine the effective dose based on these considerations that are customary in the art. As used herein, "effective dose" "Dose)" means the same thing as "effective dose".

[0058] Therefore, the effective dose is the amount that improves the clinical symptoms of the subject. Thus, as a non-limiting example, the effective dose of stem cells is sufficient to achieve the following outcomes in a subject: extubation within 28 days of receiving the first dose of MAPC cells; having PaO2 / FiO2 > 300 mmHg with PEEP < 5 cm H2O by day 28; a mortality rate ≥ 10% lower compared to subjects who did not receive MAPC (this may be determined based on past averages); or having an average increase of ≥ 4 VFDs by day 28 compared to subjects who did not receive MAPC (this may be determined based on past averages).

[0059] An "effective route" generally refers to a route that provides delivery of a drug to a desired compartment, system, or location. For example, an effective route is a route through which the drug can be administered in order to deliver a sufficient amount of the drug to produce a beneficial or desired clinical outcome at the desired site of action.

[0060] The term "exogenous," when used in relation to stem cells, generally refers to stem cells that are external to the subject and are exposed to (e.g., in contact with) an island intended for transplantation via an effective pathway. Exogenous stem cells may originate from the same subject or from different subjects. In one embodiment, exogenous stem cells may include stem cells that have been harvested from a subject, isolated, expanded ex vivo, and then exposed to an island intended for transplantation via an effective pathway.

[0061] The use of the terms "includes" is not intended to be restrictive.

[0062] "Increase" or "increasing" means to induce an overall biological event or to increase the degree of that event.

[0063] The term "isolated" refers to one or more cells that are not associated with one or more other cells, or with one or more cellular components that associate with the aforementioned one or more cells in vivo. "Enriched population" means a relative increase in the number of cells of a desired type in vivo or in primary culture compared to one or more other cell types.

[0064] However, as used herein, the term “isolated” does not indicate the presence of the cells of the present invention alone. Rather, the term “isolated” indicates that the cells of the present invention have been removed from their natural tissue environment and are present at a higher concentration compared to a normal tissue environment. Thus, an “isolated” cell population may contain, in addition to the cells of the present invention, further cell types and further tissue components. This can also be expressed, for example, with respect to cell doubling. Cells may undergo 10, 20, 30, 40 or more doublings in vivo or ex vivo, and as a result, they are enriched compared to their original number in vivo or in their original tissue environment (e.g., bone marrow, peripheral blood, placenta, umbilical cord, umbilical cord blood, etc.).

[0065] "MAPC" is an acronym for "Multipotent Adult Progenitor Cell." It refers to cells that are neither embryonic stem cells nor germ cells, but possess several of these characteristics. MAPCs can be characterized by many different descriptions, each of which gave the cells novelty at the time they were discovered. Thus, they can be characterized by one or more of these descriptions. Firstly, they can have long-term replication ability in culture, are not transformed (tumor-forming), and have a normal karyotype. Secondly, they can produce cell offspring of more than one germ layer (e.g., two or all three germ layers, i.e., endoderm, mesoderm, and ectoderm) during differentiation. Thirdly, although they are neither embryonic stem cells nor germ cells, they can express markers for these primitive cell types, so MAPCs can express one or more of Oct 3 / 4 (i.e., Oct 4, Oct 3A). Fourthly, like stem cells, they can regenerate, i.e., have long-term replication ability without transformation. This means that these cells express telomerase (i.e., have telomerase activity). Thus, the cell type referred to as "MAPC" can be characterized by alternative fundamental features that describe the above cells, through some of its novel properties.

[0066] In MAPC, the term "adult" is non-limiting. It refers to non-embryonic somatic cells as described above. MAPCs have a normal karyotype and do not form teratomas in vivo. This acronym was first used in U.S. Patent No. 7,015,037 to describe cells isolated from bone marrow that have broad replication capacity and express pluripotency markers.

[0067] MAPC represents an earlier progenitor cell population than MSCs (Verfaillie, CM, Trends Cell Biol 12:502-8 (2002), Jahagirdar, BN, et al., Exp Hematol, 29:543-56 (2001); Reyes, M. and CM Verfaillie, Ann NY Acad Sci, 938:231-233 (2001); Jiang, Y. et al., Exp Hematol, 30896-904 (2002); and Jiang, Y. et al., Nature, 418:41-9 (2002)).

[0068] The term "MultiStem®" is the trade name for a cell preparation based on MAPC (i.e., non-embryonic stem, non-germinal cells as described above) as defined in U.S. Patent No. 7,015,037. MultiStem® is prepared according to the cell culture methods disclosed in this patent application (particularly those involving lower oxygen and higher serum). MultiStem® is highly scalable, has a normal karyotype, and does not form teratomas in vivo. It can differentiate into more than one germ layer cell lineage and may express telomerase.

[0069] A "pharmaceutically acceptable carrier" is any pharmaceutically acceptable medium for cells and / or islands used in the present invention. Such a medium may maintain isotonicity, cellular metabolism, pH, etc. It is suitable for administration to a subject and can therefore be used for island and / or cell delivery and treatment.

[0070] "Progenitor cells" are cells generated during the differentiation of stem cells that possess some (but not all) of the characteristics of their terminally differentiated offspring. Defined progenitor cells (e.g., "cardiac progenitor cells") are related to a lineage, but not to a specific or terminally differentiated cell type. The term "progenitor," as used in the acronym "MAPC," does not limit these cells to a particular lineage. Progenitor cells can form more highly differentiated offspring cells than the aforementioned progenitor cells.

[0071] The term "reduce," as used herein, means to prevent and to decrease. In the context of treatment, "reduce" means either to prevent or improve a defect. This includes, but is not limited to, the reduction of the above endpoint parameters, including inflammatory biomarkers (IFNγ, IL1β, IL6, IL8, TNFα, CXCL10, IL10, MCP1, IL-1, IL-2RA, IL-7, RAGE, PD1, IL1-R2) measured over the first seven days after initial cell administration.

[0072] "Selecting" cells with a desired level of potency can mean identifying, isolating, and expanding the cells (as determined by assay). This can create a population of cells that are more potent than the parental population from which the selected cells were isolated. The "parental" cell population refers to the parental cells from which the selected cells divide. "Parent" refers to the actual P1→F1 relationship (i.e., progeny cells). Therefore, cell X is a mixed population of cell X and cell Y (where X is the expressor), If Y is isolated from another organism (which is not X), a simple isolate of X is not classified as having enhanced expression. However, if the progeny cells of X express a higher amount, those progeny cells are classified as having enhanced expression.

[0073] Selecting cells that achieve a desired effect involves both an assay to determine whether the cells achieve the desired effect and obtaining those cells. The cells may spontaneously achieve the desired effect in that the effect is not achieved by an exogenous transgene / DNA. However, effective cells may be improved by incubation with or exposure to a drug that enhances the effect. The cell population from which effective cells are selected may not be known to have the efficacy before the assay is performed. Since the effect may depend on gene expression and / or secretion, selection may be based on one or more of the genes that cause the effect.

[0074] Selection can be from cells within a tissue. For example, in this case, cells are isolated from the desired tissue, expanded in culture, and selected for achieving the desired effect, and the selected cells are further expanded.

[0075] The selection may also be from cells in ex vivo (e.g., cells in culture). In this case, one or more of the cells in the culture may be assayed for achieving the desired effect, and the obtained cells that achieve the desired effect may be further expanded.

[0076] Cells can also be selected for their enhanced ability to achieve a desired effect. In this case, the cell population from which the enhanced cells are obtained already possesses the desired effect. The enhanced effect means a higher average amount per cell compared to the parent population.

[0077] The parent population from which enhanced cells are selected may be substantially homogeneous (same cell type). One way to obtain such enhanced cells from this population is to create a single cell or cell pool, assay those cells or cell pools to obtain clones that naturally possess the enhanced (higher) effect (as opposed to treating the aforementioned cells with regulators that induce or increase the effect), and then expand those naturally enhanced cells.

[0078] However, cells may be treated with one or more agents that induce or enhance the above effects. Thus, a substantially homogeneous population may be treated to enhance the above effects.

[0079] If the above population is not substantially homogeneous, it is preferable that the parent cell population to be treated contains at least 100 of the desired cell type for which an enhanced effect is sought, more preferably at least 1,000 of the above cells, and even more preferably at least 10,000 of the above cells. After treatment, this subpopulation can be recovered from the heterogeneous population by known cell selection techniques and, if desired, further expanded.

[0080] Therefore, the desired level of effect may be higher than the level in a given preceding population. For example, cells transferred from tissue to primary culture, expanded, and isolated under culture conditions not specifically designed to produce the above effect may provide a parent population. Such a parent population may be treated to enhance the average effect per cell, or screened for one or more cells within the population that express a greater degree of effect without the intended treatment. Such cells may then be expanded to provide a population with higher (desired) expression.

[0081] "Self-renewal" of stem cells refers to the ability of a replicating daughter stem cell to produce replicating daughter stem cells that have the same differentiation potential as the original stem cell. A similar term used in this context is "proliferation."

[0082] "Subject" refers to a vertebrate (e.g., a mammal, e.g., a human). Examples of mammals include, but are not limited to, humans, dogs, cats, horses, cows, and pigs.

[0083] The term “therapeutically effective dose” refers to the amount of a drug determined to produce any therapeutic response in a mammal. For example, an effective therapeutic agent may prolong a patient’s survival and / or inhibit an obvious clinical symptom. Within the scope of the above term as used herein, a therapeutically effective treatment includes treatments that improve the quality of life of a subject, even if the treatment does not improve the disease outcome itself. Such a therapeutically effective dose is readily verifiable by those skilled in the art. Thus, “to treat” means to deliver such a dose. Thus, treating may prevent or improve any pathological symptom. In one context, treatment means improving lung function as measured by PaO2, i.e., improving toward or within the normal range (in this case, compared with an untreated patient – ​​via past averages).

[0084] In the context of this invention, the therapeutically effective amount is the amount of stem cells that produce an improvement in clinical outcomes.

[0085] The term "therapeutic time" can refer to the time required to achieve clinical improvement.

[0086] The therapeutically effective time can also refer to the time required for the subject to achieve an improved clinical state. In this case, cells may be delivered within 48 hours of the diagnosis of ARDS and evaluated over a period of 28 days.

[0087] The term "therapeutically effective route" refers to a route of administration that may be effective in achieving improved clinical outcomes. This includes intravenous delivery via either a peripheral or central line.

[0088] The appropriate amount of stem cells to achieve beneficial effects is determined empirically. The dose range may be 900,000–1,200,000 stem cells, with repeat doses potentially delivered 72–96 hours after the first dose. Therefore, these amounts must be determined empirically based on factors such as the delivery method and the severity of the disease.

[0089] The terms “treat,” “treating,” or “treatment” are used broadly in connection with the present invention, and each such term encompasses, among other things, preventing, improving, inhibiting, or curing defects, dysfunctions, diseases, or other harmful processes (including those that interfere with and / or result from treatment).

[0090] "Sufficient time" and "sufficient amount" are synonymous with "effective time" and "effective amount," respectively. Clinical symptoms that can be evaluated to determine whether the cells covered by this application are administered in a sufficient amount, for a sufficient amount of time, and through an effective route include one or more of the following: a. Reduction of pneumonia; Cytokine reduction induced by bT cells and macrophages; Reduction of chemokines involved in the recruitment of cT cells and monocytes / macrophages; increase of CD4 and CD8 T cells; d. Reduction in lymphopenia (including reduction in T cell exhaustion marker levels, reduction in T cell activation marker expression, reduction in apoptosis signaling pathways, reversal of T cell exhaustion, reduction in T cell apoptosis, increase in CD8 T cell count, increase in regulatory T cells, reduction in monocytes / macrophages in the lungs, phenotypic changes in peripheral blood monocytes, reduction in intermediate and non-classical monocytes, differentiation of regulatory T cells, proliferation of AT2 cells, reduction in pro-inflammatory cytokine production, reduction in the expression of pro-inflammatory markers, increase in M2 anti-inflammatory monocyte / macrophage differentiation, and clearance of pathogens and cellular debris in lung tissue); e. Other parameters that can be measured to determine the effectiveness of the above cells in treating the patient (including mortality, ventilator-free days, changes in consecutive organ failure, ICU-free days, oxygenation levels, oxygenation index, peak and plateau pressures, and changes from baseline in PEEP requirements (which may be compared to an untreated population)); quality of life reported by the patient (or their representative) (e.g., via survey tools such as EQ-50) may also be evaluated. f. Other parameters include decreased pulmonary vascular permeability, reduced hypoxemia and bilateral opacities on chest imaging, reduced non-cardiogenic pulmonary edema, reduced respiratory failure, positive airway pressure, inspired oxygen concentration, arterial oxygen partial pressure, and positive end-expiratory pressure; imaging scoring systems to assess the degree of pulmonary hardening, infiltration, bronchiectasis, fibrosis, or functional lung capacity; g. Other parameters include the reduction of cytokine storm syndrome.

[0091] "Verification" means confirmation. In the context of this invention, it is confirmed that cells have the desired potency to have a beneficial effect on a subject. This is to allow for a reasonable prediction of efficacy so that the cells can be used (in treatment, banking, drug screening, etc.). Thus, verification means confirming that cells that were originally found to have / established to have the desired activity actually retain that activity. Accordingly, verification is a confirmation event in a two-event process, including the initial decision and the follow-up decision. The second event is referred to herein as "verification".

[0092] The present invention may preferably be carried out using stem cells from vertebrate species (e.g., humans, non-human primates, domesticated animals, livestock, and other non-human mammals). These include, but are not limited to, the cells described below.

[0093] Transcription factor Many transcription factors and exogenous cytokines that affect the potency of stem cells in vivo have been identified. The first transcription factor to be explained as being involved in stem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit-Oct-Unc) family of transcription factors and is a DNA-binding protein that can activate gene transcription, containing an octameric sequence called an "octamer motif" within the promoter or enhancer region. Oct4 is expressed at the cleavage stage of the fertilized zygote until the oviduct is formed. The function of Oct3 / 4 is to repress differentiation-inducing genes (i.e., FoxaD3, hCG) and activate pluripotency-promoting genes (FGF4, Utf1, Rex1). Sox2 (a member of the high-mobility group (HMG) box transcription factors) works in cooperation with Oct4 to activate the transcription of genes expressed in the inner cell mass. It is essential that Oct3 / 4 expression in embryonic stem cells be maintained between certain levels. Overexpression or downregulation of Oct4 expression levels >50% alters embryonic stem cell fate, respectively, during the formation of primitive endoderm / mesoderm or trophectoderm. In vivo, Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they differentiate along the extraembryonic trophoblast lineage. Sall4 (the mammalian Spalt transcription factor) is an upstream regulator of Oct4 and is therefore important for maintaining appropriate levels of Oct4 during the early stages of development. When Sall4 levels drop below a certain threshold, trophectoderm cells ectopically expand into the inner cell mass. Another transcription factor required for pluripotency is Nanog, named after the Celtic "Tir Nan Og": Land of Eternal Youth. In vivo, Nanog is expressed from the compacted morula stage, then established in the inner cell mass, and downregulated by implantation. Downregulation of Nanog may be important to avoid uncontrolled expansion of pluripotent cells and to enable multiphyletic differentiation during gastrulation. Nanog null embryos (isolated on day 5.5) consist of a disordered blastocyst that mainly contains extraembryonic endoderm and lacks a recognizable epiblastoid layer.

[0094] Isolation and growth of MAPC Methods for isolating MAPCs are known in the art; see, for example, U.S. Patent No. 7,015,037. These methods, along with the characterization (phenotype) of MAPCs, are incorporated herein by reference. MAPCs can be isolated from a number of sources, including but not limited to bone marrow, placenta, umbilical cord and umbilical cord blood, muscle, brain, liver, spinal cord, blood, or skin. Thus, it is possible to obtain bone marrow aspirates, brain biopsies or liver biopsies, and other organs and isolate these cells using positive or negative selection techniques available to those skilled in the art, relying on genes expressed (or not expressed) in these cells (for example, by functional or morphological assays such as those disclosed in the above-mentioned applications, which are incorporated herein by reference).

[0095] MAPC also refers to Breyer et al., Experimental Hematology, 34:1596-1601 (2006); Subramanian et al. (S. These results are obtained by modified methods described in Ding (ed.), Methods Mol. Biol., 636:55-78 (2010); Boozer et al., J. Stem Cells 4(1):17-28 (2009); and Vaes et al., Methods Mol. Biol., 1235:49-58 (2015) (these methods are referenced).

[0096] MAPC derived from human bone marrow as described in U.S. Patent No. 7,015,037 MAPC does not express either CD45 or glycophorin-A (Gly-A). A mixed population of cells was subjected to Ficoll Hypaque separation. The cells were then subjected to negative selection using anti-CD45 antibody and anti-Gly-A antibody to deplete populations of CD45+ and Gly-A+ cells, and then approximately 0.1% of the remaining bone marrow mononuclear cells were recovered. The cells were also plated in wells coated with fibronectin and cultured as described below for 2-4 weeks to deplete CD45+ and Gly-A+ cells. In the culture of adherent bone marrow cells, many adherent stromal cells undergo replicative senescence at about 30 cell doublings, and a more homogeneous population of cells continues to expand and maintain long telomeres.

[0097] Further culture methods In further experiments, the density at which MAPC is cultured can vary from about 100 cells / cm 2 or about 150 cells / cm 2 to about 10,000 cells / cm 2 (from about 200 cells / cm 2 to about 1500 cells / cm 2 to about 200 cells / cm 2 and including). The density can vary between species. Further, the optical density can vary depending on the culture conditions and the source of the cells. Determining the optical density for a given set of culture conditions and cells is within the skill of those in the art.

[0098] Also, an effective atmospheric oxygen concentration of less than about 10% (including about 1-5% and particularly 3-5%) can be used at any time during the isolation, growth, and differentiation of MAPC in culture.

[0099] Cells can be cultured at various serum concentrations (e.g., about 2-20%). Fetal bovine serum can be used. Higher serum can be used in combination with a lower oxygen pressure (e.g., about 15-20%). The cells do not need to be selected prior to adhesion to the culture dish. For example, after Ficoll gradient, the cells can be, for example, 250,000-500,000 / cm 2They can be plated directly. Adhesive colonies can be picked up, pooled as much as possible, and expanded.

[0100] In one embodiment used in the experimental procedure described in the examples, high serum (approximately 15-20%) and low oxygen (approximately 3-5%) conditions were used for cell culture. Specifically, adherent cells from colonies were plated in 18% serum and 3% oxygen (containing PDGF and EGF) at a rate of approximately 1700-2300 cells / cm³. 2 It was passed through generations at this density.

[0101] In embodiments specific to MAPCs, the supplement is a cellular factor or component that enables MAPCs to retain the ability to differentiate into more than one embryonic lineage cell type (e.g., all three lineages). This may be indicated by the expression of a specific marker for the undifferentiated state (e.g., Oct 3 / 4 (Oct 3A)) and / or a marker for high expansion capacity (e.g., telomerase).

[0102] Serum can be a source of significant variation. Optimal serum concentrations can vary depending on serum batch characteristics. Therefore, different serum lots are screened for their ability to support optimal MAPC expansion. A large volume of serum from a suitable batch can be secured. Ideally, MAPC should be 200–2,000 cells / cm³. 2 They are sown at densities between [values ​​omitted], and higher densities can be avoided. They are constantly subcultivated below confluence (30-70%). Using these conditions, MAPC can conventionally be extended up to 15-20 subculturing (50-70 population doubling).

[0103] Pharmaceutical preparations In a particular embodiment, the cell population is contained within a composition that is adapted and suitable for delivery, i.e., physiologically compatible.

[0104] In some embodiments, the purity of the cells for administration with or to the islands is about 100% (substantially homogeneous). In other embodiments, it is 95% to 100%. In some embodiments, it is 85% to 95%. In particular, in the case of mixtures with other cells, the percentage may be about 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95%. Alternatively, isolation / purity may be expressed in terms of the cell doubling the cells have undergone (e.g., 10 to 20, 20 to 30, 30 to 40, 40 to 50 or more cell doublings).

[0105] Administration The dose (i.e., cell count) for humans or other mammals can be determined by those skilled in the art from this disclosure, the documents cited herein, and the knowledge of the art without excessive experimentation. The optimal dose to be used according to various embodiments of the present invention depends on many factors, including: the disease being treated and its stage; the donor's species, their health status, sex, age, weight, and metabolic rate; the donor's immunocompromise; other treatments being administered; and potential complications predicted from the donor's history or genotype. Other parameters include: whether the cells are syngeneic, autologous, allogeneic, or heterogeneous; their potency; the site and / or distribution to be targeted; and such site characteristics such as the accessibility of the cells. Further parameters include co-administration with other factors (e.g., growth factors and cytokines). The optimal dose in a given situation also takes into account how the cells are formulated, how they are administered (e.g., by injection, intraorgan, etc.), and the extent to which the cells are located at the target site after administration. [Examples]

[0106] Further details regarding the isolation and expansion of MAPC can be found above.

[0107] The population for this trial consists of men and women aged 18–89 years with a diagnosis of moderate to severe ARDS, as defined by the Berlin definition. All diagnostic criteria for moderate to severe ARDS are confirmed to be present within a 24-hour period. A 48-hour period begins after the last ARDS diagnostic criterion is met, during which infusion of MultiStem® or placebo is permitted. Subjects are confirmed to have persistent or worsening ARDS (confirmed by PaO2 / FiO2 measured within 6 hours prior to randomization). All subjects receive either MultiStem® treatment (900 million or 1.2 billion cells / dose) or placebo via intravenous (IV) infusion within 48 hours of the last ARDS diagnostic criterion being met.

[0108] The cohort was registered as follows: *Cohort 1 →Cohort 1a (MultiStem® product): Three subjects treated openly with 900 million MultiStem® cells / dose. →Cohort 1b (MultiStem® product): Three subjects treated openly with 1.2 billion MultiStem® cells / dose. →Cohort 1c (MultiStem® product): Three subjects treated open-label with 900 million or 1.2 billion MultiStem® cells / dose. The above doses were selected by DSMB based on a review of data from cohorts 1a and 1b. →Cohort 1d (MultiStem® product): Three (or more) subjects treated open-label with 900 million or 1.2 billion MultiStem® cells / dose. Subjects meeting specific criteria for persistent or worsening ARDS are eligible to receive the same second dose of 900 million or 1.2 billion MultiStem® cells / dose 72–96 hours after the initial dose, provided they continue to meet the pre-administration selection criteria. Cohort 1d will continue to replenish study subjects until at least three eligible subjects have received two doses of MultiStem®. *Cohort 2 (MultiStem® product induction phase (Run-In phase)): 50 subjects randomized in a 1:1 ratio to either MultiStem® treatment (900 million or 1.2 billion cells / dose) or placebo. *Cohort 3 (MultiStem® product): 300-400 subjects randomized in a 1:1 ratio to receive either MultiStem® treatment (900 million or 1.2 billion cells / dose) or placebo.

[0109] In cohorts 1d, 2, and 3, subjects with persistent or worsening ARDS could receive a second dose of MultiStem® or placebo identical / equal to the first dose administered (900 million or 1.2 billion cells / dose or equivalent placebo) 72–96 hours after the initial dose of MultiStem® or placebo, provided they continued to meet the pre-administration selection criteria; no crossovers were observed. Specific study visits for data collection included day 0 (pre-infusion and post-infusion), as well as days 1, 2, 3, 7, 14, 21, 28, 60, 90, 180, and 365. For subjects receiving the second dose of IP, data collection also included days 4, 5, and 6. The data evaluated include adverse events (AEs), vital signs, safety laboratory parameters (biochemistry, hematology, and coagulation), COVID-19 test results, respiratory physiological measures and ventilator settings (PaO2 / FiO2 ratio, oxygenation index, peak and plateau pressure, PEEP, and subject position information [i.e., prone, supine, or inclined]), quality of life (EuroQoL Five Dimension Questionnaire [EQ-5D]-5L), hospitalization data (days on mechanical ventilation, days in the intensive care unit [ICU], and days hospitalized), mortality, Continuing Organ Failure Assessment (SOFA) score, and surveyed biomarkers (leukocyte population and inflammatory biomarkers). In certain embodiments, for example, the following are provided: (Item 1) A method for treating virus-induced acute respiratory distress syndrome (ARDS), the method comprising the step of administering to a subject having virus-induced ARDS a sufficient amount, for a sufficient duration, and by an effective route, wherein the cells (I) are non-embryonic stem non-germinoid cells that have undergone cell duplication of at least 10-40 times in culture, the cells (I) express telomerase and / or oct4, are not transformed, are not tumorigenic, and have a normal karyotype. (Item 2) The cell (I) expresses telomerase, as described in item 1. (Item 3) The method according to item 1 or 2, wherein the cell (I) can differentiate into at least two of the endoderm, ectoderm, and mesoderm cell types. (Item 4) The cell (I) expresses oct4, according to any of the methods described in items 1 to 3. (Item 5) The cell (I) is human, according to any of the methods described in items 1 to 4. (Item 6) The cells (I) are derived from bone marrow and are obtained using any of the methods described in items 1 to 5. (Item 7) The cell(I) has undergone 40 cell doubling in culture, according to any one of items 1 to 6. (Item 8) The cell(I) is capable of undergoing at least 40 cell duplication in culture, according to any one of items 1 to 7. (Item 9) The cell(I) is an allogene, as described in any of items 1 to 8. (Item 10) The method according to any one of items 1 to 9, wherein the virus that induced the aforementioned ARDS is Betacoronavirus. (Item 11) The method according to item 10, wherein the Betacoronavirus is selected from the group consisting of severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (Item 12) The method according to item 1, wherein the virus is selected from the group consisting of influenza, coronaviruses including (SARS-CoV, MERS-CoV, SARS-CoV2), herpes simplex virus, cytomegalovirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, and adenovirus. (Item 13) The subject is a human, and the method is one of items 1 to 12. (Item 14) The method according to any one of items 1 to 13, wherein the route of administration is intravenous.

Claims

[Claim 1] The invention described in the specification.