Stem cells for treating respiratory diseases

Abstract

The present invention relates to the use of integrin alpha 10 selected mesenchymal stem cells in the treatment of airway disorders, diseases and trauma affecting the airways.

Description

[Technical Field]

[0001] The present invention relates to integrin alpha 10 selected mesenchymal stem cells and their use in the treatment of respiratory diseases. [Background technology]

[0002] There is a significant unmet clinical need for treating airway disorders such as acute respiratory distress syndrome (ARDS), a severe respiratory failure characterized by the rapid onset of widespread inflammation in the lungs. ARDS has a significant adverse effect on critical care patients and remains a serious clinical disorder with a high mortality rate (estimated at approximately 40%) and can result in significantly higher levels of long-term morbidity among surviving patients.

[0003] Symptoms include shortness of breath, respiratory distress, and bluish skin discoloration. ARDS can be caused by direct or indirect causes, depending on whether the lungs are initially compromised. Direct causes include pneumonia (including bacterial or viral infections (e.g., COVID-19)), aspiration, inhalation lung injury, pulmonary contusion, chest trauma, and drowning. Indirect causes include sepsis, shock, pancreatitis, trauma, cardiopulmonary bypass, and burns (Matthay, 2019). A rare case of ARDS has been associated with the administration of large volumes of fluid during resuscitation after trauma (Casay, 2019).

[0004] ARDS is typically triggered by acute injury; this acute injury triggers a powerful immune response and the release of numerous immune mediators, known as a cytokine storm, which can adversely affect multiple organs (e.g., the lungs) (Gonzales, 2015). ARDS is characterized by excessive activation of the immune system, which causes severe damage to lung tissue, making specific drugs targeting specific molecules or pathways of limited effectiveness. Recent advances in ARDS management have been largely symptomatic, aiming to preserve respiratory gas exchange, thereby sustaining life while physicians wait for the underlying disease to resolve (Prescott, 2016). These strategies include protective mechanical ventilation, neuromuscular blockade, prone positioning, and the use of fluid balance management strategies. However, the latter is not a practical treatment for ARDS because it merely aims to preserve respiratory gas exchange. To further reduce mortality, treatment of ARDS should target the inflammatory mechanisms underlying lung injury. However, no pharmacological therapies currently exist that can effectively affect disease-specific pathways or reduce mortality, thus necessitating novel treatments for ARDS and related disorders. Summary of the Invention

[0005] The present inventors have found that integrin alpha 10-selected mesenchymal stem cells (MSCs) improve hemodynamic stability and oxygen supply, and reduce blood clot formation and lung tissue damage in an ARDS animal model. Furthermore, MSCs exhibit specific immunomodulatory and anti-inflammatory properties. Therefore, integrin alpha 10-selected MSCs are particularly suitable for alleviating the adverse effects of respiratory diseases (such as ARDS and related pulmonary complications).

[0006] In one main aspect, the present invention relates to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in the treatment of one or more diseases or trauma of the respiratory system / airways of a mammal; and / or in connection with transplantation of one or more organs or tissues of the airways of a mammal.

[0007] In a further aspect, the present disclosure is directed to a method of treating a respiratory disease, disorder, or injury in a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0008] In a further aspect, the present disclosure is directed to a method for transplantation therapy or promotion of an organ or tissue of the respiratory tract in a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0009] In a further aspect, the present disclosure is directed to a method of preventing blood clotting in a mammal in association with a disease, disorder, or trauma to the respiratory system of the mammal and / or in association with transplantation of an organ or tissue of the respiratory tract of the mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0010] In a further aspect, the present disclosure is directed to a method of promoting hemodynamic stability in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, the method comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0011] In a further aspect, the present disclosure is directed to a method of reducing the need for inotropic therapy in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, the method comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0012] In a further aspect, the present disclosure is directed to a method of improving oxygenation in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0013] In a further aspect, the present disclosure is directed to a method of preventing tissue damage, e.g., preventing structural tissue damage, in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0014] In a further aspect, the present disclosure is directed to a method of reversing tissue damage, e.g., a method of reversing structural tissue damage, in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0015] In a further aspect, the present disclosure is directed to a method of reducing neutrophil counts in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0016] In a further aspect, the present disclosure is directed to a method of increasing lymphocyte numbers in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0017] In a further aspect, the present disclosure is directed to a method of reducing inflammatory cytokines in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0018] In a further aspect, the present disclosure is directed to a method of increasing interferon alpha in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0019] In a further aspect, the present disclosure is directed to a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs), for use in any of the following methods in connection with a disease, disorder, or trauma of the respiratory system of a mammal and / or in connection with transplantation of an organ or tissue of the respiratory tract of a mammal: In mammals: ·How to prevent blood clotting; ·Methods to promote hemodynamic stability; ·Methods to reduce the need for inotropic therapy; ·Methods to improve oxygen supply; · A method for preventing tissue damage, for example a method for preventing structural damage to tissue; · Methods for reversing tissue damage, e.g., methods for reversing structural damage to tissue; ·Methods to reduce neutrophil counts; ·How to increase lymphocyte count; ·Methods to reduce inflammatory cytokines; and / or ·How to increase interferon alpha. [Brief explanation of the drawings]

[0020] [Figure 1]Reduced need for inotropic therapy in MSC-treated animals. ARDS was induced in 12 pigs, who were treated intravenously with either integrin alpha 10-selected MSCs (6 pigs) or a cryopreservation solution containing DMSO (6 pigs). Inotropic therapy, such as norepinephrine administration, is used in ARDS models to ensure hemodynamic stability and oxygen delivery levels throughout the course of the experiment. Therefore, the amount of norepinephrine administered serves as a measure of the animal's hemodynamic stability and ability to deliver oxygen to the lungs. Compared to the control group (untreated), MSC-treated animals (treated) required significantly less total norepinephrine. This indicates that animals treated with integrin alpha 10-selected MSCs had more stable hemodynamics and better oxygen distribution. **** indicates p ≤ 0.001. [Figure 2] Improvement of oxygen delivery capacity with MSC treatment. Twelve hours after the start of treatment, oxygen delivery capacity analysis was possible in three of the MSC-treated pigs and three of the control pigs. Oxygen delivery capacity was measured by analyzing the ratio between arterial oxygen partial pressure (PaO2) and inspired oxygen (FIO2). The results show that oxygen delivery capacity was improved in the MSC-treated pigs (treated) (PaO2 / FIO2 ratio: 21-28) compared with the control pigs (untreated) (PaO2 / FIO2 ratio: XX). This suggests that lung structure for gas exchange is better preserved in the MSC-treated animals compared with the control animals. [Figure 3]Prolonged clotting time in MSC-treated animals. Because coagulation (blood clotting) is a key feature of ARDS and contributes to an extreme inflammatory response, we investigated clotting time in an ARDS model. Clotting time was observed in both MSC-treated (circles) and untreated (squares) groups. This graph shows clotting time (seconds) at different stages of the experiment, demonstrating a prolonged clotting time in the MSC-treated group (circles). The effect in the treated group was already evident 2 hours after infusion of integrin alpha 10-selected MSCs and persisted for at least 12 hours. The prolonged clotting time correlated with the alleviation of ARDS, demonstrating the therapeutic efficacy and safety of integrin alpha 10-selected MSCs as an ARDS therapy. [Figure 4] Reduced lung tissue damage after MSC treatment. To investigate the degree of lung tissue damage and the effect of MSCs on lung tissue structure, lung biopsies were performed from the upper (upper lobe), middle (middle lobe), and lower (lower lobe) lungs at the end of the ARDS study. The results showed that lung tissue damage was milder in MSC-treated animals compared with control (untreated) animals, demonstrating the therapeutic effect of MSCs in the ARDS model. Representative images shown in the top row are from the untreated group, and representative images shown in the bottom row are from the MSC-treated group. All images were taken at 20x magnification (black scale bar represents 0.1 mm; white scale bar represents 0.2 mm). [Figure 5]Reduction of neutrophils in the blood of MSC-treated animals. (A) To determine the effect of integrin α10-selected MSCs on inflammation in ARDS pigs, we analyzed the number of neutrophils in the blood. Results showed a decrease in neutrophil counts after MSC infusion (treatment), indicating that fewer neutrophils were recruited to the lung tissue of the treatment group as part of the inflammatory response. This suggests that neutrophil reduction is an important mechanism of action of MSCs, thereby reducing the degree of inflammation and lung tissue damage. Mean ± standard error. p ≤ 0.05. (B) The graph shows the lymphocyte counts (million cells per ml) in the animals' peripheral blood. Briefly, in the treatment group, the number of lymphocytes in whole blood was elevated at the end of the experiment, peaking at 9 and 10 hours after infusion. Lymphocytes are key immune cells involved in the response to ARDS, and a high lymphocyte count may be an indicator of less severe ARDS cases. [Figure 6]Plasma and bronchoalveolar lavage fluid (BALF) cytokines support the immunomodulatory effects of MSCs. To further explore the mechanism of action of the immunomodulatory effects of integrin alpha 10-selected MSCs in an ARDS model, blood samples and bronchoalveolar lavage fluid (BALF) were collected at multiple time points during the experimental course and analyzed for several inflammatory cytokines involved. Herein, we demonstrate the effects of MSCs on interferon alpha (IFN-α) and interleukins IL-12, IL-1β, and IL-6, supporting the immunomodulatory effects in MSC-treated animals. (A) Compared with untreated animals, treated animals showed higher levels of the inflammatory cytokine interferon alpha (IFN-α) in the plasma 1 hour after infusion of integrin alpha 10-selected MSCs, which persisted for an additional 3 hours. These higher levels may suggest a more favorable prognosis for ARDS, as evidenced by a recent retrospective study showing that infusion of IFN-α reduces mortality in ARDS patients. (B) Compared with control (untreated) animals, animals treated with integrin alpha 10-selected MSCs showed lower plasma levels of the inflammatory cytokine interleukin (IL)-12 1 hour after MSC infusion, suggesting an immediate effect of MSCs, which persisted for 8 hours. This further supports the immunomodulatory effect of MSCs, as this may be an indicator of a lower number of activated antigen-presenting cells in the blood. (C) and (D) After completion of the study (endpoint), MSC-treated animals had lower concentrations of the inflammatory interleukins IL-1β and IL-6 (considered to be important factors in the development of ARDS) in the BALF compared with untreated animals. Furthermore, both IL-1β and IL-6 significantly increased compared to baseline in untreated animals only. This may suggest the presence of macrophages polarized toward an inflammatory state (M1), and overall lung inflammation was reduced in treated animals. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition The terms "anti-integrin alpha10 antibody" or "anti-integrin alpha10 subunit antibody" used herein are synonyms for antibodies capable of recognizing and binding to at least the integrin alpha10 subunit of the heterodimeric protein integrin alpha10beta1. These antibodies may be antibodies that recognize an epitope of the heterodimeric protein integrin alpha10beta1, where the epitope includes amino acid residues of both the integrin alpha10 and integrin beta1 subunits.

[0022] As used herein, "integrin alpha10" or "integrin alpha10" refers to the alpha10 subunit of the heterodimeric protein integrin alpha10beta1. This term does not exclude the presence of an integrin beta1 subunit that binds to the integrin alpha10 subunit to form the quaternary structure of the integrin alpha10beta1 heterodimer. The sequence of the human integrin alpha10 chain is known and is published in GenBank™ / EBI databank under accession number AF074015, and is also described in Camper (1998). "Alpha" and "alpha," as well as "alpha10" and "alpha10," are synonyms.

[0023] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] The term "some embodiments" may include one or more embodiments.

[0025] As used throughout this specification, the words "a" or "an," or when used in connection with the term "comprising" in the Claims and / or this specification, may mean "one," but is also consistent with the meanings "one or more," "at least one," and "one or more than one."

[0026] As used herein, the terms "isolating," "sorting," and "selecting" refer to the act of identifying cells as a particular type of cell and separating them from cells that do not belong to the same cell type or from cells in other differentiation states. These terms can also refer to the act of identifying cells by the presence of a particular marker. For example, in the present invention, integrin alpha 10-selected mesenchymal stem cells (MSCs) are the subject of the present invention. Isolation generally refers to the first step of separation, which may be, for example, mechanical separation; on the other hand, "selection" is more specific and is performed, for example, by using antibodies. Those skilled in the art will understand that the process of "isolating," "sorting," or "selecting" cells results in enrichment of the cells.

[0027] The term "integrin alpha 10 enriched MSCs" used herein refers to the term "integrin alpha 10 高 MSCs,” “Integrin alpha 10 selected mesenchymal stem cells” and “Integrin alpha 10 in mesenchymal stem cells” 高As described in Example 1, MSCs for use in the present invention are selected by a process that enriches for MSCs that express integrin alpha 10; for example, MSCs that express integrin alpha 10 are selected using an antibody that specifically binds to integrin alpha 10. Cells selected for a specific property, for example, MSCs that express integrin alpha 10 or MSCs that express integrin alpha 10, can be selected. 高 Those skilled in the art will appreciate that MSCs can form specific homogeneous cell populations.

[0028] As used herein, "mesenchymal stem cells" or "MSCs" refer to multipotent stromal cells as defined by the Committee on Mesenchymal and Tissue Stem Cells of the International Society for Cellular Therapy (see Dominici, M. et al., Cell Therapy (8(4):315-7(2006))). When maintained under standard culture conditions, MSCs must adhere to plastic; express CD105, CD73, and CD90, but not CD45, CD34, CD14 or CD11b, CD79alpha or CD19, and HLA-DR surface molecules. MSCs must have the ability to differentiate in vitro into osteoblasts, adipocytes, or chondroblasts.

[0029] As used herein, the term "respiratory system disease, disorder, or injury" refers to any malfunction of one or more parts of the respiratory system in the system involved in breathing. The respiratory system (also called the airways, respiratory tract, or ventilation system) includes, for example, the lungs, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli (sometimes called the lower respiratory tract), as well as the trachea, larynx, pharynx, nasal cavity, and paranasal sinuses (sometimes called the upper respiratory tract).

[0030] The term "ARDS (acute respiratory distress syndrome)" as used herein refers to life-threatening inflammation, often accompanied by pulmonary edema leading to severe respiratory failure. ARDS is a clinical syndrome of lung injury resulting from hypoxic respiratory failure caused by intense (often widespread) lung inflammation following severe physiological injury.

[0031] The term "sepsis" as used herein refers to a condition defined as "systemic inflammatory response syndrome (SIRS) secondary to infection." Such conditions are characterized by infectious symptoms caused by microorganisms, preferably bacteria or fungi, parasites, or viruses or prions. The term "sepsis" as used herein includes sepsis associated with the final stages of sepsis, as well as the development of "severe sepsis," "septic shock," and "sepsis complications" (e.g., multiple organ dysfunction syndrome (MODS), disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), and acute renal failure (AKI)), and includes all stages of sepsis.

[0032] As used herein, "preventing" or "prevention" includes delaying or stopping the onset of a disease, disorder, or condition.

[0033] The terms "disease," "disorder," "injury," and "syndrome" herein, as well as other similar terms (such as "pathological condition"), may be understood as synonyms within this disclosure and refer to a non-functional, pathological, non-physiological, and / or disordered state.

[0034] Disease indication In one aspect, the present disclosure is directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in the treatment of a disease, disorder, or trauma of the respiratory system of a mammal, and / or in connection with organ or tissue transplantation of the respiratory tract of a mammal.

[0035] In some embodiments of the present disclosure, the respiratory disease is a lower respiratory tract disease.

[0036] In some embodiments of the present disclosure, the respiratory disease is a respiratory disease that primarily affects the lung interstitium.

[0037] In some embodiments of the present disclosure, the respiratory disease is a respiratory disease that affects the airways.

[0038] In some embodiments of the present disclosure, the respiratory disease is a disease that primarily affects the pulmonary interstitium, selected from the group consisting of acute respiratory distress syndrome (ARDS), pulmonary edema, pulmonary eosinophilia, idiopathic interstitial pneumonia, primary interstitial lung disease specific to infancy or childhood, interstitial lung disease associated with systemic disease, alveolar microlithiasis, lymphangioleiomyomatosis, and lipoid pneumonia.

[0039] As shown in the examples, the integrin alpha 10-selected MSCs disclosed herein exhibit anti-inflammatory and immunomodulatory properties and can be used to ameliorate, prevent, and / or treat ARDS-related symptoms. Those skilled in the art will appreciate that diseases or disorders associated with ARDS can also be treated with the integrin alpha 10-selected MSCs disclosed herein. For example, diseases or disorders associated with ARDS (such as COVID-19-associated cytokine release syndrome (CRS), cytokine storm syndrome (CSS), and multisystem inflammatory syndrome); or complications adversely affecting newborns (e.g., premature infants) may adversely affect the respiratory system (e.g., lungs) and may be treated with the integrin alpha 10-selected MSCs disclosed herein. Cytokine storm and cytokine release syndrome are life-threatening systemic inflammatory syndromes associated with elevated circulating cytokine levels and immune cell hyperactivation, which can be caused, for example, by various treatments, pathogens, cancer, autoimmune conditions, and monogenic diseases.

[0040] In some embodiments of the present disclosure, the respiratory disease is acute respiratory distress syndrome (ARDS) and / or related disorders.

[0041] In some embodiments of the present disclosure, the respiratory disease is ARDS.

[0042] A respiratory disease, disorder or syndrome that may be treated using the compositions disclosed herein may be, for example, a disorder associated with ARDS.

[0043] Thus, in some embodiments of the present disclosure, the respiratory disease is cytokine release syndrome (CRS).

[0044] In some embodiments of the present disclosure, the respiratory disease is cytokine storm syndrome (CSS).

[0045] In some embodiments of the present disclosure, the ARDS-related disease is multisystem inflammatory syndrome associated with COVID-19.

[0046] In some embodiments of the present disclosure, the respiratory disease is cytokine-mediated ARDS.

[0047] In some embodiments of the present disclosure, the respiratory disease is neonatal ARDS / respiratory distress syndrome.

[0048] In some embodiments of the present disclosure, the respiratory disease is neonatal respiratory distress (such as neonatal respiratory distress syndrome).

[0049] 10. A composition for use according to any one of the preceding claims, wherein the respiratory disease is ARDS due to trauma.

[0050] In some embodiments of the present disclosure, the respiratory disease is ARDS caused by a viral or bacterial infection. For example, viral infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause ARDS.

[0051] It is understood that ARDS can have multiple causes. However, regardless of the cause, ARDS is considered a clinical syndrome defined by clinical parameters, such as severe hypoxemia despite supplemental oxygen, bilateral pulmonary infiltrates, and reduced lung compliance. Multiple measurable factors (e.g., cytokines) are thought to be or are known to be involved in the development of ARDS. Those skilled in the art will understand that integrin alpha 10-selected mesenchymal stem cells (MSCs) can be used to treat ARDS caused by different types of causes. Although the examples described herein describe commonly accepted animal models of ARDS, those skilled in the art will understand that other models known in the art can also be used to evaluate the efficacy of the MSCs disclosed herein.

[0052] Sepsis is a frequent cause of ARDS, and ARDS is often the condition that ultimately leads to death in septic patients. Therefore, the integrin alpha 10-selected mesenchymal stem cells (MSCs) disclosed herein are effective in treating ARDS, and may be used to treat the cause of ARDS (e.g., sepsis).

[0053] Another cause of ARDS is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) / COVID-19, and ARDS can be a condition that ultimately leads to death in patients with COVID-19. Therefore, the integrin alpha 10-selected mesenchymal stem cells (MSCs) disclosed herein are effective in treating ARDS and may be used to treat the cause of ARDS (e.g., COVID-19).

[0054] That is, in some embodiments of the present disclosure, the respiratory disease is ARDS caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) / COVID-19.

[0055] In some embodiments of the present disclosure, the respiratory disease is ARDS due to other causes.

[0056] The disease mechanisms involved in ARDS (such as increased inflammatory cytokines and cellular changes, particularly to immune cells) may also be involved in mechanisms involved in the rejection of transplanted organs or tissues (e.g., in the case of lung transplants). Thus, compositions comprising the integrin alpha 10-selected mesenchymal stem cells (MSCs) disclosed herein may be used to prevent or treat disorders associated with organ transplants (e.g., lung transplants).

[0057] That is, some embodiments of the present disclosure relate to the use of compositions comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) in the treatment of diseases, disorders, or trauma associated with organ or tissue transplantation (e.g., lung transplantation) of the mammalian airway.

[0058] MSC characterization and production In some embodiments of the present disclosure, at least 60% of the MSCs express the integrin alphal0 subunit.

[0059] Example 1 describes a method for producing integrin alpha10-selected MSCs presented in this disclosure. A major advantage of integrin alpha10-selected MSCs is that the selected MSCs are homogeneous cultures and / or populations because they are selected based on the expression of integrin alpha10 protein. These cells have been shown to stably express stem cell markers (see, e.g., WO2018 / 138322). Those skilled in the art will appreciate that multiple cell selection methods are available for enriching cells. In the present invention, integrin alpha10 高 MSCs are enriched by an isolation / selection procedure. In this regard, an anti-integrin alpha 10 antibody may be used. MSC isolation and selection may be performed by the method described in WO2018 / 138322.

[0060] As disclosed in Example 1, in the selection step, anti-integrin alpha 10 antibody is used, and the "selected MSCs" selected based on the cellular integrin alpha 10 expression are those that express integrin alpha 10 (MSCs that express integrin alpha 10 are referred to as integrin alpha 10 MSCs). 高 (Sometimes referred to as MSCs). More specifically, the integrin alpha10 subunit is expressed together with the integrin beta1 subunit, so the selected cells are MSCs that express the heterodimer integrin alpha10 beta1 (α10β1). This selection step is followed by an expansion step, during which the integrin alpha10 expression of each selected MSC may vary; that is, not all MSCs may express integrin alpha10 at all times during the expansion process, even at the time of administration. However, at the time of administration of the MSCs to a patient, at least 50% of the administered cells express the integrin alpha10 subunit.

[0061] In some embodiments of the present disclosure, at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, at least 100% etc. of the MSCs express the integrin alpha 10 subunit.

[0062] In some embodiments of the present disclosure, the MSCs are negative for MHC class II, CD45, CD34, CD11b and / or CD19.

[0063] In some embodiments of the present disclosure, the MSCs express CD73, CD90 and / or CD105.

[0064] In some embodiments of the present disclosure, the MSCs are selected from the group consisting of mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal stromal cells, or a mixture thereof.

[0065] In some embodiments of the present disclosure, the MSCs are induced to express the integrin alphal0 subunit.

[0066] In some embodiments of the present disclosure, the MSCs are cultured in a medium comprising mammalian serum and FGF-2.

[0067] In some embodiments of the present disclosure, the MSCs are cultured in a medium containing platelet lysate and / or components of platelet lysate.

[0068] In some embodiments of the present disclosure, the MSCs are cultured in a medium comprising FGF-2 and platelet lysate and / or a component of platelet lysate.

[0069] In some embodiments of the present disclosure, the MSCs are cultured in a medium comprising mammalian serum and platelet lysate and / or a component of platelet lysate.

[0070] In some embodiments of the present disclosure, the MSCs are cultured in a medium containing TGFβ.

[0071] In some embodiments of the present disclosure, the MSCs are cultured in a medium comprising FGF2.

[0072] In some embodiments of the present disclosure, the MSCs are cultured in serum-free medium containing platelet lysate and / or components of platelet lysate.

[0073] In some embodiments of the present disclosure, the MSCs are cultured in serum-free medium containing growth factors.

[0074] In some embodiments of the present disclosure, the MSCs are cultured in serum-free medium containing the growth factors FGF2 and / or TGF.

[0075] In some embodiments of the present disclosure, the MSCs are allogeneic or autologous cells.

[0076] In some embodiments of the present disclosure, the MSCs and the mammal are from the same species.

[0077] In some embodiments of the present disclosure, the MSCs and the mammal are from different species.

[0078] In some embodiments of the present disclosure, the MSCs are derived from adipose tissue, bone marrow, synovium, peripheral blood, cord blood, umbilical cord blood, Wharton's gel, and / or amniotic fluid.

[0079] Those skilled in the art will appreciate that the integrin alphal0 selected MSC selection methods disclosed herein can also be used to select MSCs from other sources known in the art.

[0080] In some embodiments of the present disclosure, the MSCs are derived from adipose tissue.

[0081] In some embodiments of the present disclosure, the MSCs are derived from bone marrow.

[0082] In some embodiments of the present disclosure, the MSCs are derived from fetal, neonatal, juvenile or adult MSCs and / or progenitor cells.

[0083] In some embodiments of the present disclosure, the MSCs are not derived from embryonic cells or embryos.

[0084] In some embodiments of the present disclosure, the MSCs are in vitro cell culture.

[0085] In some embodiments of the present disclosure, the selection of the MSCs is performed using an anti-integrin alphal0 antibody.

[0086] Administration In some embodiments of the present disclosure, the composition comprising the integrin alphal0 selected MSCs is administered to the lung or airways.

[0087] In some embodiments of the present disclosure, the composition comprising the integrin alphal0 selected MSCs is administered by injection.

[0088] Those skilled in the art will be aware of other methods for administering the integrin alphal0 selected MSCs that are known in the art.

[0089] In some embodiments of the present disclosure, the composition comprising the integrin alphal0 selected MSCs is administered parenterally.

[0090] Thus, compositions comprising the integrin alphal0 selected MSCs used as described in the present disclosure may be administered topically for the purpose of penetrating any mucous membrane of the animal to which the integrin alphal0 selected MSCs are administered.

[0091] In some embodiments of the present disclosure, the composition comprising the integrin alphal0 selected MSCs is administered by intravenous injection, intramuscular injection and / or intratracheal instillation, or any combination thereof.

[0092] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs are formulated as cell aggregates prior to administration.

[0093] In some embodiments of the present disclosure, the composition comprising the integrin alphal0 selected MSCs is administered in the form of a cell suspension together with a pharmaceutically acceptable excipient.

[0094] In some embodiments of the present disclosure, compositions comprising the integrin alpha 10 selected MSCs are administered during surgery to repair damaged lungs.

[0095] In some embodiments of the present disclosure, the composition comprising the integrin alpha 10 selected MSCs is administered in connection with a lung transplant.

[0096] In some embodiments of the present disclosure, the mammal is a human.

[0097] In some embodiments of the present disclosure, the mammal is a human, horse, pony, bull, donkey, mule, camelid, cat, dog, pig, or cow.

[0098] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs and the mammal are from the same species.

[0099] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs and the mammal are from different species.

[0100] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs are derived from adipose tissue, bone marrow, synovium, peripheral blood, cord blood, umbilical cord blood, Wharton's gel, and / or amniotic fluid.

[0101] In some embodiments of the present disclosure, the integrin alpha 10 selected MSCs are derived from adipose tissue.

[0102] In some embodiments of the present disclosure, the integrin alpha 10 selected MSCs are derived from bone marrow.

[0103] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs are derived from fetal, neonatal, juvenile or adult MSCs and / or progenitor cells.

[0104] In some embodiments of the present disclosure, the integrin alpha 10 selected MSCs are not derived from embryonic cells or embryos.

[0105] In some embodiments of the present disclosure, the integrin alphal0 selected MSCs are in vitro cell culture.

[0106] In some embodiments of the present disclosure, selection of the integrin alphal0 selected MSCs is performed using an anti-integrin alphal0 antibody.

[0107] In some embodiments of the present disclosure, the composition comprising the integrin alpha 10 selected mesenchymal stem cells (MSCs) further comprises an anti-inflammatory agent and / or an immunomodulatory agent.

[0108] In another aspect, the present disclosure is directed to the use of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs) for the manufacture of a therapeutic medicament relating to the treatment of a disease, disorder or trauma of the respiratory system of a mammal, and / or organ or tissue transplantation of the respiratory tract of a mammal.

[0109] method In a further aspect, the present disclosure is directed to a method of treating a disease, disorder, or injury of the respiratory system in a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0110] In a further aspect, the present disclosure is directed to a method for transplantation therapy or promotion of an organ or tissue of the respiratory tract in a mammal, the method comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0111] In a further aspect, the present disclosure is directed to a method of preventing blood clotting in a mammal in association with a disease, disorder, or trauma to the respiratory system of the mammal and / or in association with transplantation of an organ or tissue of the respiratory tract of the mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0112] In a further aspect, the present disclosure is directed to a method of promoting hemodynamic stability in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, the method comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0113] In a further aspect, the present disclosure is directed to a method of reducing the need for inotropic therapy in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, the method comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0114] In a further aspect, the present disclosure is directed to a method of improving oxygenation in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0115] In a further aspect, the present disclosure is directed to a method of preventing tissue damage, e.g., preventing structural tissue damage, in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0116] In a further aspect, the present disclosure is directed to a method of reversing tissue damage, e.g., a method of reversing structural tissue damage, in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0117] In some embodiments of the present disclosure, the tissue damage prevented or reversed by the methods of the present disclosure is lung tissue damage.

[0118] In some embodiments of the present disclosure, the tissue damage prevented or reversed by the methods of the present disclosure is interstitial tissue damage, alveolar septal damage, airway damage, vasculature damage, and / or nervous system damage.

[0119] In a further aspect, the present disclosure is directed to a method of reducing neutrophil counts in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0120] In a further aspect, the present disclosure is directed to a method of increasing lymphocyte numbers in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0121] In a further aspect, the present disclosure is directed to a method of reducing inflammatory cytokines in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alphal0-selected mesenchymal stem cells (MSCs).

[0122] In some embodiments of the present disclosure, the inflammatory cytokines that are reduced in the methods of the present disclosure are selected from the group consisting of interleukin-12 (IL-12), IL-1β, IL-6, and IL-4, or any combination thereof.

[0123] In some embodiments of the present disclosure, the inflammatory cytokines that are reduced in the methods of the present disclosure are reduced in the blood and / or bronchoalveolar lavage fluid.

[0124] In a further aspect, the present disclosure is directed to a method of increasing interferon alpha in association with a disease, disorder, or trauma of the respiratory system of a mammal and / or in association with organ or tissue transplantation of the respiratory tract of a mammal, comprising administering a therapeutically effective amount of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0125] In a further aspect, the present disclosure is directed to a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs), for use in any of the following methods: In connection with disease, disorder, or trauma of the mammalian respiratory system and / or in connection with transplantation of organs or tissues of the mammalian respiratory tract; In mammals: ·How to prevent blood clotting; ·Methods to promote hemodynamic stability; ·Methods to reduce the need for inotropic therapy; ·Methods to improve oxygen supply; · A method for preventing tissue damage, for example a method for preventing structural damage to tissue; · Methods for reversing tissue damage, e.g., methods for reversing structural damage to tissue; ·Methods to reduce neutrophil counts; ·How to increase lymphocyte count; ·Methods to reduce inflammatory cytokines; and / or ·How to increase interferon alpha.

[0126] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of preventing blood clotting in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's respiratory tract.

[0127] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of promoting hemodynamic stability in a mammal in connection with a disease, disorder, or trauma of the mammal's respiratory system and / or in connection with transplantation of an organ or tissue of the mammal's airway.

[0128] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of reducing the need for inotropic therapy in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's airway.

[0129] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of improving oxygenation capacity in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's airway.

[0130] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of preventing tissue damage in a mammal, e.g., a method of preventing structural tissue damage, in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's respiratory tract.

[0131] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method for reversing tissue damage in a mammal, e.g., a method for reversing structural tissue damage, in connection with a disease, disorder, or trauma of the mammal's respiratory system and / or in connection with transplantation of an organ or tissue of the mammal's airway.

[0132] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of reducing neutrophil counts in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's respiratory tract.

[0133] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of increasing lymphocyte numbers in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's respiratory tract.

[0134] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of reducing inflammatory cytokines in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's airway.

[0135] Some embodiments of the present disclosure are directed to a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in a method of increasing interferon alpha in a mammal in association with a disease, disorder, or trauma of the mammal's respiratory system and / or in association with transplantation of an organ or tissue of the mammal's respiratory tract.

[0136] In some embodiments of the present disclosure, the tissue damage is lung tissue damage.

[0137] In some embodiments of the present disclosure, the tissue damage is interstitial tissue damage, alveolar septal damage, airway damage, vasculature damage, muscle damage, and / or nervous system damage.

[0138] 70. The method of claim 69, wherein the inflammatory cytokine is selected from the group consisting of interleukin 12 (IL-12), IL-1β, IL-6 and IL-4, or any combination thereof.

[0139] In some embodiments of the present disclosure, the inflammatory cytokines are reduced in the blood and / or bronchoalveolar lavage fluid.

[0140] Example Example 1: Preparation of integrin alpha 10 enriched MSCs the purpose This example describes methods for isolating, selecting, expanding, and storing integrin alpha 10-selected MSCs for use in therapeutic models.

[0141] Materials and Methods Integrin alpha 10-selected mesenchymal stem cells (MSCs) were isolated from human or animal adipose donor tissue or other sources containing MSCs. The adipose tissue was disaggregated / digested, and the adipose-derived stromal vascular fraction (SVF) was resuspended in MSC growth medium and then seeded into cell culture flasks to allow MSCs to adhere to the plastic and proliferate.

[0142] The plastic-adherent cells were analyzed by flow cytometry for expression of cell surface markers CD73, CD90, and CD105 (≥95%) and absence of expression of CD45, CD34, CD11b, CD19, and HLA-DR (≤2%). These specific antigen expression criteria are also part of the MSC definition proposed by the International Society for Cellular Therapy (Dominici, 2006). MSC preparations were grown in monolayer culture in MSC growth medium, and integrin α10-expressing MSCs were selected using an antibody that specifically binds to integrin α10 (which recognizes the intact receptor integrin α10 beta 1, i.e., integrin α10β1); selection was performed by magnetic bead separation or FACS cell sorting. The integrin α10-selected MSCs were further expanded to confirm cell surface expression of defined MSC antigens and their potential for hematopoietic differentiation. The α10-selected MSCs were live-frozen in cryopreservation medium and cryopreserved until use.

[0143] result The above process yielded integrin alpha 10 selected MSCs, which were expanded and frozen into vials that could be used for administration, for example, intravenous administration.

[0144] conclusion The manufacturing process described above produces alpha10 selected MSCs that meet the minimum standards defining human MSCs and may be applicable to cell therapy.

[0145] Example 2: Demonstration of efficacy and safety of integrin alpha 10-selected MSC treatment in a porcine ARDS model the purpose The objective of these experiments was to demonstrate and evaluate the therapeutic effect of integrin alpha 10-selected MSCs on ARDS as well as the safety of intra-arterial infusion of integrin alpha 10-selected MSCs in a validated porcine model.

[0146] Materials and Methods Twelve pigs (mean weight 35.83 ± 4.79 kg) were used. General anesthesia was administered, and a peripheral intravenous catheter was placed in the earlobe. Simultaneous endotracheal intubation and mechanical ventilation with non-humidified air were also performed. Ventilation was adjusted to maintain carbon dioxide levels (PaCO2) between 33 and 41 mmHg.

[0147] To induce ARDS, lipopolysaccharide (LPS) derived from the gram-negative bacterium Escherichia coli was diluted and administered via intratracheal (ET) and pulmonary artery.

[0148] Hemodynamics, gas exchange, inotropic therapy requirements, fluid replacement requirements, urine output, cytokine responses, and plasma coagulation cascade responses were continuously monitored throughout the study.Lung tissue was collected for RNA sequencing and immunohistochemical analysis.

[0149] To confirm and monitor the porcine ARDS model and evaluate the clinical efficacy of infused integrin alpha 10-selected MSCs, hemodynamic parameters, blood gases, and blood clotting time (using a ROTEM device) were measured. The different stages of ARDS were determined according to the Berlin definition, which uses the ratio of partial pressure of oxygen (PaO2) to the fraction of inspired oxygen (FIO2). After the establishment of ARDS, pigs were randomized to receive either treatment with integrin alpha 10-selected MSCs (5 million MSCs / kg, administered intravenously) or a sham-treated cryotherapy medium containing 5–10% DMSO. At the end of the experiment, lung biopsies were taken from the entire right lobe via sternotomy. Hematoxylin and eosin-stained lung biopsies were used to confirm the occurrence of severe lung injury and the efficacy of infused MSCs in preserving lung integrity in our model.

[0150] result Analysis of hemodynamic stability / need for inotropic therapy To ensure hemodynamic stability and oxygen supply levels during the course of the ARDS model, inotropic therapy, such as norepinephrine administration, was performed. Compared to the control group, animals treated with integrin alpha10-selected MSCs showed a significant reduction in the total amount of administered norepinephrine, indicating more stable hemodynamic parameters and better oxygen distribution in these animals (Figure 1).

[0151] Analysis of oxygen supply capacity Concerning the reduction in norepinephrine dose, blood gas values ​​also confirmed the increased oxygenation capacity in ARDS animals treated with integrin alpha10-selected MSCs. After 12 hours, it was possible to analyze the oxygenation capacity of three integrin alpha10-selected MSC-treated and three control animals. Compared to control animals, animals treated with integrin alpha10-selected MSCs showed improved oxygenation capacity, supporting the therapeutic effect of integrin alpha10-selected MSCs (Figure 2).

[0152] Clotting time analysis Blood coagulation and clot formation are common and significant medical problems in ARDS. Therefore, we investigated the effect of integrin alpha 10-selected MSCs infused during the course of an ARDS study on clotting (blood clotting) time. Compared to control animals, integrin alpha 10-selected MSC-treated animals showed significantly reduced clotting time. Extended The effect was already apparent after 2 hours, and was significant after 3 hours (Fig. 3). extension is an important efficacy parameter and demonstrates the safety of intravenously administered integrin alpha 10-selected MSCs. These results support that infusion of integrin alpha 10-selected MSCs is an effective treatment for preventing clot formation in ARDS, one of the main hallmarks of this condition (Frantzeskaki, 2017).

[0153] histology Hematoxylin and eosin staining of lung tissue sections taken from the upper, middle, and lower lobes of the lungs was analyzed to compare the lung histology between pigs treated with integrin alpha 10-selected MSCs and untreated pigs. While severe lung tissue damage was observed in control animals, the integrin alpha 10-selected MSC-treated group showed significantly less lung tissue damage and significantly better preserved lung structure (Figure 4). These results are consistent with the clinical findings regarding hemodynamics and oxygen delivery, clearly demonstrating the therapeutic efficacy of integrin alpha 10-selected MSCs in this ARDS model.

[0154] conclusion The safety and efficacy of integrin alpha 10-selected MSCs in an animal model were established by comparing pigs treated with integrin alpha 10-selected CS with pigs treated with a placebo. In a clinically relevant porcine ARDS model, integrin alpha 10-selected MSCs were found to be effective in treating ARDS. Intravenously administered integrin alpha 10-selected MSCs improved hemodynamics and pulmonary oxygenation, reduced blood clot formation, and preserved the integrity of lung tissue architecture. Example 3: Demonstration of anti-inflammatory and immunomodulatory effects of integrin alpha 10-selected MSCs in a porcine ARDS model the purpose To investigate the mechanism of action of integrin alpha 10-selected MSCs in a porcine ARDS model, e.g., to examine their anti-inflammatory and / or immunomodulatory effects.

[0155] Materials and Methods Blood samples were collected from integrin alpha 10-selected MSC-treated and control pigs at multiple time points during the ARDS study to analyze plasma neutrophil counts and concentrations of multiple pro- and anti-inflammatory cytokines. Cytokine levels were analyzed in plasma and bronchoalveolar lavage (BAL) samples at multiple time points using a multiplex immunoassay kit measuring nine cytokines. Analysis of the immunophenotype of peripheral blood mononuclear cells (PBMCs) at multiple time points reflects cytokine profiles and may correlate with clinical outcomes over the course of the study. Lung biopsies could also be used to gain insight into pathophysiology and compartment infiltrating cells. Bronchoalveolar lavage fluid (BALF) samples were also collected at the beginning and end of the study. Neutrophil and lymphocyte counts were analyzed using a Sysmex, and concentrations of multiple cytokines were measured using a Luminex multiplex assay with cytokine-specific antibodies.

[0156] result Analysis of neutrophil and lymphocyte counts Neutrophil and lymphocyte counts were analyzed in blood samples at multiple time points during the ARDS study. Compared with control animals, MSC-treated animals had lower neutrophil counts, suggesting the immunomodulatory and anti-inflammatory effects of integrin alpha 10-selected MSCs. Differences between animals treated with integrin alpha 10-selected MSCs and untreated animals were observed even after 1 hour, and the difference increased over time (Figure 5A). Analysis of lymphocyte counts revealed an increase in lymphocytes 8 hours after treatment in integrin alpha 10-selected MSC-treated animals, but not in untreated animals (Figure 5B). This finding is supported by a retrospective analysis of ARDS patients showing that higher lymphocyte counts correlate with improved survival (Song, 2020).

[0157] Inflammatory cytokine analysis of blood and BALF samples Compared to control animals, animals treated with integrin alpha10-selected MSCs had lower plasma levels of several inflammatory cytokines, including interleukin-12 (IL-12), IL-1β, and IL-6. This difference was observed as early as 1 hour after infusion of integrin alpha10-selected MSCs, suggesting an immediate immunomodulatory effect of integrin alpha10-selected MSCs. Compared to control animals, animals treated with integrin alpha10-selected MSCs had lower plasma levels of the inflammatory cytokine interleukin (IL)-12 even 1 hour after MSC infusion, suggesting an immediate effect of MSCs that persists for at least 6 hours (Figure 6B). This may be indicative of fewer activated antigen-presenting cells present in the blood (Dorman, 2000), further supporting the immunomodulatory effect of MSCs.

[0158] Furthermore, compared to untreated animals, integrin alpha 10-selected MSC-treated animals had elevated levels of interferon alpha (IFN-α) at ​​1 hour and persisted for several hours after infusion of integrin alpha 10-selected MSCs (Figure 6A). Interestingly, elevated IFN-α levels have been shown to be associated with a better prognosis in patients with ARDS (Wang, 2020).

[0159] At the end of the study, cytokine levels in BALF were analyzed. Results showed that BALF levels of the pro-inflammatory cytokines IL-1β (IL-1b) and IL-6 were significantly increased in untreated animals compared to treated animals (Figure 6C, D). This may suggest fewer inflammatory-polarized macrophages and overall reduced lung inflammation (McGonagle, 2020).

[0160] By examining inflammatory markers and immune response profiles in treated and untreated animals, important insights into the anti-inflammatory mechanisms or immunomodulatory effects of integrin alpha 10-selected MSCs were gained. It will be appreciated that the results of this study, described in Examples 2 and 3, can be confirmed in larger pigs (e.g., 60-70 kg pigs) and in other established models of ARDS and related disorders.

[0161] conclusion In a porcine model of severe ARDS, we demonstrated that infusion of integrin alpha10-selected MSCs resulted in a reduction in blood neutrophil counts and reduced levels of inflammatory cytokines in plasma and BALF. This suggests that integrin alpha10-selected MSCs have anti-inflammatory and immunomodulatory effects in this ARDS model, which may reflect the mechanisms of action responsible for the hemodynamic improvement and preservation of lung integrity observed in integrin alpha10-selected MSC-treated animals. The lower levels of circulating cytokines in plasma in integrin alpha10-selected MSC-treated animals suggest a lower risk of developing the cytokine storm (Hojyo, 2000), a hallmark of ARDS, and also indicate a less severe progression of ARDS.

[0162] References Camper, Hellman, Lundgren-Akerlund; J Biol Chem. 1998 Aug 7;273(32):20383-9; Isolation, cloning, and sequence analysis of the integrin subunit alpha10, a beta1-associated collagen binding integrin expressed on chondrocytes. Casey, Semler, Rice; Semin Respir Crit Care Med. 2019 Feb;40(1):57-65; Fluid Management in Acute Respiratory Distress Syndrome. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F. C., and Krause, D. S. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8, 315-317 . Dorman, Holland; Cytokine Growth Factor Rev. 2000 Dec;11(4):321-33; Interferon-gamma and interleukin-12 pathway defects and human disease. Frantzeskaki, Armaganidis, Orfanos; Respiration. 2017;93(3):212-225. doi: 10.1159 / 000453002. Epub 2016 Dec 21, Immunothrombosis in Acute Respiratory Distress Syndrome: Cross Talks between Inflammation and Coagulation. Gonzales, Lucas, Verin; Austin J Vasc Med. 2015 Jun 4;2(1):1009; The Acute Respiratory Distress Syndrome: Mechanisms and Perspective Therapeutic Approaches. Hojyo, Uchida, Tanaka, Hasebe, Tanaka, Murakami and Hirano; Cytokine Growth Factor Rev. 2000 Dec;11(4):321-33; Interferon-gamma and interleukin-12 pathway defects and human disease. Matthay; Nat Rev Dis Primers. 2019 Mar 14;5(1):18; Acute respiratory distress syndrome. McGonagle, Sharif , O’Regan, Bridgewood; Autoimmun Rev; 2020 Jun;19(6):102537. doi: 10.1016 / j.autrev.2020.102537. The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Prescott; Am J Respir Crit Care Med. 2016 Jul 15;194(2):147-55; Toward Smarter Lumping and Smarter Splitting: Rethinking Strategies for Sepsis and Acute Respiratory Distress Syndrome Clinical Trial Design. Song, Liu, Lu, Luo, Peng, Chen; BMC Pulm Med. 2020 Apr 23;20(1):102; Prognostic factors for ARDS: clinical, physiological and atypical immunodeficiency. Wang, N. et al. Retrospective multicenter cohort study shows early interferon therapy is associated with favorable clinical responses in COVID-19 patients. Cell Host Microbe https: / / doi.org / 10.1016 / j.chom.2020.07.005 (2020).

[0163] item Item 1: Enriched integrin α10 高 A composition comprising a population of mesenchymal stem cells (MSCs), for use in connection with the treatment of one or more diseases or trauma of the respiratory system of a mammal and / or the transplantation of one or more organs or tissues of the respiratory tract of a mammal. Item 2: The composition of item 1, wherein the respiratory disease is a respiratory disease that primarily affects the lung interstitium. Item 3: A composition for use according to any one of the preceding items, wherein the respiratory disease is acute respiratory distress syndrome (ARDS) and related disorders. Item 4: A composition for use according to any one of the preceding items, wherein the respiratory disease is ARDS. Item 5: A composition for use according to any one of the preceding items, wherein the respiratory disease is cytokine release syndrome (CRS). Item 6: A composition for use according to any one of the preceding items, wherein the respiratory disease is cytokine storm syndrome (CSS). Item 7: A composition for use according to any one of the preceding items, wherein the respiratory disease is cytokine-mediated ARDS. Item 8: A composition for use according to any one of the preceding items, wherein the respiratory disease is neonatal ARDS / respiratory distress syndrome. Item 9: A composition for use according to any one of the preceding items, wherein the respiratory disease is ARDS caused by trauma. Item 10: A composition for use according to any one of the preceding items, wherein the respiratory disease is ARDS caused by a viral or bacterial infection. Item 11: A composition for use according to any one of the preceding items, wherein the respiratory disease is ARDS caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) / COVID-19. Item 12: A composition for use according to any one of the preceding items, wherein the respiratory disease is ARDS due to other causes. Item 13: A composition used in accordance with any one of the preceding items, wherein at least 60% of the cells of the MSC population express the integrin alpha 10 subunit. Item 14: A composition for use according to any one of the preceding items, wherein at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100%, of the total cells comprised in the enriched mesenchymal stem cell (MSC) population express the integrin alpha 10 subunit. Item 15: A composition used according to any one of the preceding items, wherein the MSCs are MHCII-negative and / or CD45-negative. Item 16: A composition used in accordance with any one of the preceding items, wherein the MSCs express CD44, CD90 and CD105. Item 17: A composition for use in accordance with any one of the preceding items, wherein the MSCs are selected from the group consisting of mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal stromal cells; or a mixture thereof. Item 18: A composition for use according to any one of the preceding items, wherein the cells are induced to express the integrin alpha 10 subunit. Item 19: A composition used according to any one of the preceding items, wherein the cells are cultured in a medium comprising mammalian serum and FGF-2. Item 20: A composition used according to any one of the preceding items, wherein the cells are cultured in a medium comprising a platelet lysate and / or a component of a platelet lysate. Item 21: A composition used according to any one of the preceding items, wherein the cells are cultured in a medium comprising FGF-2 and platelet lysate and / or a component of a platelet lysate. Item 22: A composition used according to any one of the preceding items, wherein the cells are cultured in a medium comprising mammalian serum and platelet lysate and / or a component of a platelet lysate. Item 23: A composition used according to any one of the preceding items, wherein the cells are cultured in a medium containing TGFβ. Item 24: A composition used according to any one of the preceding items, wherein the cells are cultured in a serum-free medium comprising platelet lysate and / or components of a platelet lysate. Item 25: A composition used according to any one of the preceding items, wherein the cells are cultured in a serum-free medium containing growth factors. Item 26: A composition used according to any one of the preceding items, wherein the cells are cultured in a serum-free medium containing the growth factors FGF2 and / or TGFβ. Item 27: A composition used according to any one of the preceding items, wherein the MSCs are allogeneic or autologous cells. Item 28: A composition for use according to any one of the preceding items, wherein the MSCs are administered to the lung or airways. Item 29: A composition used in accordance with any one of the preceding items, wherein the population of MSCs is administered by injection. Item 30: A composition used according to any one of the preceding items, wherein the population of MSCs is administered in the form of a cell suspension together with a pharmaceutically acceptable excipient. Item 31: A composition used in accordance with any one of the preceding items, wherein the population of MSCs is formulated as cell aggregates prior to administration. Item 32: A composition for use according to any one of the preceding items, wherein the population of MSCs is administered during surgery for the purpose of repairing a damaged lung. Item 33: A composition for use according to any one of the preceding items, wherein the population of MSCs is administered in connection with lung transplantation. Item 34: A composition for use according to any one of the preceding items, wherein the mammal is a human, horse, pony, bull, donkey, mule, camelid, cat, dog, pig, or cow. Item 35: A composition for use according to any one of the preceding items, wherein the mammal is a human. Item 36: A composition used according to any one of the preceding items, wherein the MSCs and the mammal are derived from the same species. Item 37: A composition used according to any one of the preceding items, wherein the MSCs and the mammal are derived from different species. Item 38: A composition for use according to any one of the preceding items, wherein the MSCs are derived from adipose tissue, bone marrow, synovium, peripheral blood, umbilical cord blood, Wharton's gel, and / or amniotic fluid. Item 39: A composition used in accordance with any one of the preceding items, wherein the MSCs are derived from adipose tissue. Item 40: The use or method according to any one of Items 76 to 85, wherein the MSCs are derived from bone marrow. Item 41: A composition for use according to any one of the preceding items, wherein the cells are derived from fetal, neonatal, juvenile or adult MSCs and / or progenitor cells. Item 42: A composition used according to any one of the preceding items, wherein the cells are not derived from embryonic cells or embryos. Item 43: A composition used according to any one of the preceding items, wherein the cell population is an in vitro cell culture. Item 44: A composition used according to any one of the preceding items, wherein the concentration is carried out using an anti-integrin alphal0 antibody. Item 45: A composition used according to any one of the preceding items, further comprising an anti-inflammatory agent.

Claims

1. A pharmaceutical composition for use in the treatment of acute respiratory distress syndrome (ARDS) and / or related disorders, comprising integrin alpha 10-selected mesenchymal stem cells (MSCs), wherein at least 60% of the MSCs in the composition express integrin alpha 10.

2. 2. The pharmaceutical composition of claim 1, wherein the acute respiratory distress syndrome (ARDS) is selected from the group consisting of cytokine-mediated ARDS, neonatal ARDS / respiratory distress syndrome, ARDS due to trauma, ARDS due to viral or bacterial infection, ARDS due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) / COVID-19, and ARDS due to other causes.

3. The pharmaceutical composition of claim 1 or 2, wherein at least 65% of the MSCs express the integrin alpha 10 subunit.

4. 4. The pharmaceutical composition of claim 1, wherein the MSCs are negative for MHC class II, CD45, CD34, CD11b and / or CD19, and / or the MSCs express CD73, CD90 and / or CD105.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the MSCs are allogeneic or autologous cells.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the composition comprising the MSCs is administered to the lung or airways.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the composition comprising the MSCs is administered by injection.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the composition comprising the MSCs is administered parenterally.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the composition comprising the MSCs is administered by intravenous injection, intramuscular injection and / or intratracheal instillation, or any combination thereof.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the composition comprising the MSCs is administered during surgery for the purpose of repairing damaged lungs.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the composition comprising the MSCs is administered in connection with lung transplantation.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the MSCs are derived from adipose tissue, bone marrow, synovium, peripheral blood, umbilical cord blood, Wharton's gel, and / or amniotic fluid.

13. In relation to acute respiratory distress syndrome (ARDS), In mammals: - a method for preventing blood clotting; - methods to promote hemodynamic stability; - a method for reducing the need for inotropic therapy; - methods for improving oxygen delivery capacity; - A method for preventing tissue damage, for example a method for preventing structural damage to tissue; - A method for reversing tissue damage, for example a method for reversing structural damage to tissue; - methods to reduce neutrophil counts; -Methods for increasing lymphocyte counts; - a method for reducing inflammatory cytokines; and / or - a method for increasing interferon alpha, 10. Use of a composition in the manufacture of a medicament for use in treating a patient with a rheumatoid arthritis, wherein the composition comprises integrin alpha 10-selected mesenchymal stem cells (MSCs), and at least 60% of the MSCs in the composition express integrin alpha 10.

Images