Constant natural killer t cells for treating acute respiratory distress syndrome (ARDS)

Abstract

The present disclosure relates, at least in part, to compositions comprising constant natural killer T (iNKT) cells (e.g., unmodified allogeneic iNKT cells), and uses of the compositions comprising the iNKT cells to treat a disease or a symptom or complication of a disease (e.g., a viral infection, a disease secondary to a primary disease (e.g., a neurological disease). The present invention relates to a method for treating acute respiratory distress syndrome (ARDS) and / or organ failure associated therewith (e.g., viral infection).

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 423,036, filed on November 6, 2022, and U.S. Provisional Application Serial No. 63 / 503,431, filed on May 19, 2023, under 35 U.S.C. 119(e), the entire contents of which are incorporated herein by reference. Background Art

[0003] Acute respiratory distress syndrome (ARDS) is a lung inflammatory syndrome with a high mortality rate. Most deaths attributable to ARDS are not due to respiratory failure but rather to progressive dysfunction of other organs, also known as multisystem organ failure (MSOF). Effective treatments to reduce or prevent ARDS and post-ARDS MSOF remain elusive. Summary of the Invention

[0004] The present disclosure relates, at least in part, to compositions comprising invariant natural killer T (iNKT) cells (e.g., unmodified allogeneic iNKT cells), and methods of using compositions comprising the iNKT cells to treat a disease or symptoms or complications of a disease (e.g., viral infection, acute respiratory distress syndrome (ARDS) secondary to a primary disease (e.g., viral infection) and / or its associated organ failure). In some embodiments, the compositions and methods provided herein reduce inflammation (e.g., lung inflammation associated with a viral infection). In some embodiments, the compositions and methods provided herein reduce secondary infections (e.g., secondary bacterial and / or fungal infections following a viral infection). In some embodiments, administration of the composition to a subject (e.g., a subject suffering from ARDS secondary to a viral infection) results in improved survival, reduced inflammatory response, reduced occurrence or severity of pneumonia, and / or reduced occurrence or severity of organ failure following ARDS.

[0005] In some aspects, the present disclosure provides a method of treating a subject having a viral infection, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

[0006] In some aspects, the present disclosure provides a method for treating a subject having acute respiratory distress syndrome (ARDS) (e.g., moderate or severe ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

[0007] In some aspects, the present disclosure provides a method for reducing organ damage or preventing organ damage in a subject at risk of organ damage, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells. In certain embodiments of the present invention, the subject at risk of organ damage suffers from acute respiratory syndrome (ARDs) and / or viral infection. In certain embodiments of the present invention, the subject with organ damage suffers from acute respiratory syndrome (ARDs) and / or viral infection.

[0008] In some aspects, the present disclosure provides a method for inducing an anti-inflammatory response in a subject having acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

[0009] In some aspects, the present disclosure provides a method of reducing or preventing concomitant infection in a subject having acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

[0010] In some aspects, the present disclosure provides a method of reducing or preventing concomitant infection in a subject receiving invasive mechanical ventilation or veno-venous extracorporeal membrane oxygenation (VV ECMO), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells. In certain embodiments, the subject receiving invasive mechanical ventilation or VV ECMO suffers from acute respiratory distress syndrome (ARDS).

[0011] In certain embodiments, the present disclosure provides a method for reducing or preventing a hospital-acquired infection in a subject at risk of a hospital-acquired infection, the method comprising administering to the subject a composition comprising constant natural killer T (iNKT cells). In certain embodiments, the subject at risk of acquiring a hospital-acquired infection suffers from a viral infection. In certain embodiments, the subject at risk of acquiring a hospital-acquired infection is receiving invasive mechanical ventilation or venovenous extracorporeal membrane oxygenation (VV ECMO). In certain embodiments of the present invention, the subject at risk of acquiring a hospital-acquired infection suffers from acute respiratory distress syndrome (ARDS).

[0012] In some embodiments, the iNKT cells are unmodified.

[0013] In some embodiments, the iNKT cells are derived from a donor who is not the subject. In some embodiments, the iNKT cells are allogeneic. In some embodiments, the iNKT cells are isolated from peripheral blood mononuclear cells and expanded ex vivo.

[0014] In some embodiments, the donor is human.

[0015] In some embodiments, at least 90% of the cells in the composition are iNKT cells. In some embodiments, at least 95% of the cells in the composition are iNKT cells.

[0016] In some embodiments, ARDS is associated with a viral infection. In some embodiments, the viral infection is caused by a coronavirus, influenza virus, enterovirus, rhinovirus, parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), or human metapneumovirus. In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), or Middle East respiratory syndrome coronavirus (MERS-CoV). In some embodiments, the influenza virus is H1N1 influenza, H5N1 influenza, or H7N9 influenza.

[0017] In some embodiments, ARDS is associated with sepsis, trauma (eg, severe trauma with shock and multiple transfusions), cardiopulmonary bypass, transfusion of blood products, and severe burns.

[0018] In some embodiments, the subject is not receiving mechanical ventilation.

[0019] In some embodiments, the subject is mechanically ventilated while being administered the composition.

[0020] In some embodiments, the subject is refractory to mechanical ventilation.

[0021] In some embodiments, the subject receives extracorporeal membrane oxygenation (ECMO). In some embodiments, the ECMO is venovenous extracorporeal membrane oxygenation (VV ECMO). In some embodiments, there is no oxygenator malfunction due to blockage.

[0022] In some embodiments, administration of the composition does not induce cytokine release syndrome.

[0023] In some embodiments, administration of the composition improves survival of the subject relative to a subject not administered the composition.

[0024] In some embodiments, administration of the composition induces an anti-inflammatory response in the subject as measured by one or more cytokines, wherein the one or more cytokines include: IL-1α / 1β, IL-6, ferritin, C-reactive protein (CRP), IL-2, IL-5, IL-7, IP-10, IL-15, IL-12p70, IFNγ, TFNα, IL-17A, IL-1RA, IL-4, IL-10, IL-13, IL-8, MCP-1, MIP-1α, VEGF or VEGF-D.

[0025] In some embodiments, administration of the composition reduces the incidence of associated infections (e.g., VAP) relative to subjects not administered the composition. In some embodiments, the associated infection is a hospital-acquired infection. In some embodiments, hospital-acquired infections include Klebsiella aerogenes, catheter-related bloodstream infections due to Candida albicans, and ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP).

[0026] In some embodiments, the administration of the composition reduces the occurrence of one or more organ failures relative to subjects not administered the composition. In some embodiments, the organ failure includes renal failure, liver failure, blood system failure and / or nervous system failure. In some embodiments, the organ failure is renal failure.

[0027] In some embodiments, 80 x 10 6 Up to 2000x10 6 In some embodiments, 100 x 10 iNKT cells are administered to a subject. 6 In some embodiments, 300 x 10 iNKT cells are administered to a subject. 6 In some embodiments, 1000 x 10 iNKT cells are administered to a subject. 6 iNKT cells.

[0028] In some embodiments, administration is via intravenous injection or intravenous infusion.

[0029] In some embodiments, the composition is administered once to the subject. In some embodiments, the subject can be administered one or more times after the initial administration.

[0030] In some embodiments, the subject is also administered dexamethasone and / or remdesivir.

[0031] In some embodiments, administration results in an improvement in the subject's lung function compared to the subject's lung function before administration. In some embodiments, administration results in an increase in the subject's lung capacity compared to the subject's lung capacity before administration. In some embodiments, administration results in an increase in the subject's lung parenchyma stability compared to the subject's lung parenchyma stability before administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments and, together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

[0033] Figure 1is a schematic diagram showing the antiviral mechanism by innate natural killer T (iNKT) cells.

[0034] Figure 2 Shown are the dates of COVID-19 diagnosis for patients in the context of the prevalence of SARS-CoV-2 strains in the United States.

[0035] Figure 3 The 30-day survival rate in the study is shown compared to the survival results of the control population. Patients in the study showed a 70% survival rate compared to a 10% survival rate in the control group.

[0036] Figures 4A to 4F It was shown that treatment with iNKT cell therapy does not induce cytokine release syndrome (CRS). Figure 4A IL-1α levels before and after iNKT cell treatment are shown. Figure 4B IL-1β levels before and after iNKT cell treatment are shown. Figure 4C IL-6 levels before and after iNKT cell treatment are shown. Figure 4D Shown are ferritin levels after iNKT cell treatment. Figure 4E Shown are C-reactive protein (CRP) levels after iNKT cell treatment. Figure 4F Shown are D-dimer levels after iNKT cell treatment.

[0037] Figure 5 The 90-day survival curve of patients undergoing venovenous extracorporeal membrane oxygenation (VV ECMO) with iNKT cell therapy is shown, compared to a control group (patients undergoing venovenous extracorporeal membrane oxygenation (VV ECMO) but not receiving iNKT cell therapy). The VV ECMO + iNKT cell therapy group showed a 75% survival rate, while the control group showed only a 30% survival rate.

[0038] Figures 6A to 6D Peripheral persistence of iNKT cells in the blood of patients on invasive mechanical ventilation (IMV) or VV ECMO was shown. Figures 6A to 6C Cohort-level peripheral persistence of iNKT cells in patient PBMCs is shown by digital PCR based on genetic markers specific to the donor material. Each cohort received a different dose of iNKT cells. Each line represents data from a single patient. Donor iNKT cells were detected until day 6 post-infusion, with possible low-level persistence being detectable at the highest dose level, persisting longer until the last sampling day on day 28. Peak iNKT cell levels demonstrated dose proportionality. Data from patients undergoing ECMO are shown in red. Figures 6A to 6B Representative results from a patient in IMV who received iNKT cells. Figure 6CIncluded are representative results from patients in IMV or VVECMO who received iNKT cells. Figure 6D The kinetics of tissue distribution of iNKT cells in a murine xenograft model are shown, demonstrating that iNKT cells rapidly translocate to tissues following intravenous (iv) injection. The observed persistence of iNKT cells in the patient's blood after a brief infusion is consistent with the kinetics of blood-to-tissue distribution of iNKT cells in vivo.

[0039] Figures 7A to 7R Representative graphs showing serum cytokine levels of selected biomarkers across the immunomodulatory spectrum. Figures 7A to 7I are representative graphs showing the production of proinflammatory cytokines after iNKT cell infusion. Figures 7L to 7M Shown is the production of anti-inflammatory cytokines after iNKT cell infusion. Figures 7N to 7P Shown is the production of chemokines after iNKT cell infusion. Figures 7Q to 7R Shown is the production of growth factors following iNKT cell infusion.

[0040] Figures 8A to 8D are representative graphs showing the presence of donor-specific alloantibodies (DSA) determined on the day of dosing and on day 14. Figures 8A to 8B Shown in HLA class I matching ( Figure 8A ) or HLA class II matching ( Figure 8B ) induction of DSA in patients. Figure 8C is a representative graph showing serum levels of DSA after dosing, which decreased with increasing degree of HLA matching. DSA levels with a combined MFI < 1,000 were considered negative and not reported. Figure 8D : is a representative graph showing DSA levels measured at discharge of four patients (patient discharge day, from left to right: 60, 28, 21, 28). DSA levels after iNKT cell infusion peaked on day 14 and appeared to decline thereafter (data were normalized to peak DSA levels for each patient).

[0041] Figures 9A to 9C are representative graphs showing survival, iNKT cell persistence, and anti-inflammatory cytokine production in patients undergoing veno-venous extracorporeal membrane oxygenation (VV-ECMO) and receiving iNKT cell therapy. Figure 9A Survival was shown to be 100% at 14 days and 75% at 30 and 90 days, with a mean ECMO run time of 133.5 days. Figure 9B The persistence of circulating iNKT cells in the venovenous ECMO cohort was shown to be comparable to that in study patients not undergoing ECMO. Figure 9C It is shown that a significant increase in the levels of the anti-inflammatory cytokine IL1-RA was observed.

[0042] Figures 10A to 10B is a chest X-ray image showing improved lung function within 24 hours after iNKT cell infusion. Figure 10A is a chest X-ray image before infusion. Figure 10B is a chest X-ray image after infusion. DETAILED DESCRIPTION

[0043] The present disclosure relates, at least in part, to compositions comprising invariant natural killer T (iNKT) cells (e.g., unmodified allogeneic iNKT cells) and methods of using compositions comprising iNKT cells to treat a disease or a symptom or complication of a disease (e.g., acute respiratory distress syndrome (ARDS) secondary to a primary disease (e.g., a viral infection). In some embodiments, the present disclosure is based on the unexpected observation that administration of iNKT cells to a subject (e.g., a subject with ARDS secondary to a viral infection) results in a decrease in the number of cells in the subject receiving 1000×10 6 In some embodiments, the invention provides a method for treating patients with ARDS who have undergone invasive mechanical ventilation (IMV) and who have received iNKT cell therapy, resulting in improved survival (e.g., 30-day survival of 70% for subjects undergoing invasive mechanical ventilation (IMV) who received iNKT cell therapy, relative to a 30-day survival of 10% for subjects undergoing IMV who did not receive iNKT cell therapy; and 90-day survival of 75% for subjects undergoing venovenous extracorporeal membrane oxygenation (VV ECMO), relative to a 90-day survival of approximately 30% for subjects undergoing VV ECMO who did not receive iNKT cell therapy), reduced inflammatory response, reduced occurrence or severity of concomitant infection and pneumonia, and / or reduced occurrence or severity of organ failure after ARDS. Furthermore, iNKT cell therapy offers at least the following benefits: (i) a favorable safety profile (e.g., no neurotoxicity or grade ≥3 TRAEs were observed); (ii) a transient persistence in the periphery, consistent with in vivo data describing rapid translocation of iNKT cells from the blood to tissues; (iii) the opportunity for repeat dosing (although alloantibodies were detected after iNKT cell administration and correlated with the degree of HLA matching, the antibody response was transient); and (iv) the ability to treat viral diseases and infections (a reduced incidence of pneumonia was observed in patients treated with the highest dose of iNKT cell therapy).

[0044] In addition, the present disclosure provides a variant agnostic method for ARDS (e.g., COVID-19 ARDS) and is the first immune cell therapy used in patients undergoing ECMO. Although there is a trend of improvement in the mortality rate of severe COVID-19 respiratory failure, mainly due to early corticosteroids and antiviral therapy, the mortality rate remains high (47.9% to 84.4%). Adding venovenous extracorporeal membrane oxygenation (VV-ECMO) support improves the survival rate of ARDS patients (e.g., ARDS in COVID-19 patients), but the overall mortality rate at 90 days is still low (i.e., about 47%). Complications of VV-ECMO therapy include bleeding, oxygenator malfunction and hospital-acquired infections, including but not limited to Klebsiella aerogenes, catheter-related bloodstream infection due to Candida albicans, and ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP). For patients with severe COVID-19 respiratory failure, multimodal therapy is needed to enhance current interventions. This publication reports the first safe administration of allogeneic human unmodified constant natural killer T (iNKT) cell infusions in patients with severe COVID-19 respiratory failure receiving venovenous extracorporeal membrane oxygenation (VV-ECMO). In contrast to previous mesenchymal stem cell therapies in patients with ARDS on ECMO, no oxygenator failures associated with this cell therapy due to filter clogging were observed.

[0045] The foregoing and other aspects, implementations, actions, functions, features, and embodiments of the present teachings may be more fully understood from the following description in conjunction with the accompanying drawings.

[0046] I. Therapeutic Treatment Using Invariant Natural Killer T (iNKT) Cells

[0047] In some aspects, the present disclosure provides a method for treating a subject with a viral infection, the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. As used herein, the term "administering" or "administration" means providing a therapeutic agent (e.g., iNKT cells) or a composition thereof (e.g., a composition comprising iNKT cells) to the subject in a physiologically and / or pharmacologically useful manner (e.g., treating the subject's disease or symptoms or complications associated with the disease). As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient suffering from or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). As used herein, the term "treating" or "treatment" refers to the application or administration of a composition comprising one or more active agents (e.g., unmodified allogeneic iNKT cells) to a subject having a target disease or condition (e.g., ARDS), symptoms or complications of a disease / condition (e.g., respiratory distress, multiple organ failure), or a susceptibility or primary indication of a disease / condition (e.g., viral infection) with the intent to cure, heal, alleviate, alleviate, alter, remedy, improve, ameliorate, or affect the condition (e.g., ARDS), symptoms or complications of a disease (e.g., respiratory distress, multiple organ failure), or a susceptibility or primary indication of a disease or condition (e.g., viral infection). Alleviating the target disease / condition includes delaying or preventing the development or progression of the disease, reducing disease severity, and / or promoting survival.

[0048] iNKT cell therapy elicits antiviral effects in at least the following ways: (i) recognition of CD1d ligands in diseased tissues and activation through constant TCR; (ii) recognition of stress signals through activating NK receptors (e.g., NKG2D, DNAM1); (iii) regulation and / or destruction of myeloid-derived suppressor cells and inflammatory monocytes; (iv) recruitment and activation of NK cells and T cells through cytokine secretion; (v) reversal of T cell exhaustion; and (vi) cytokine-mediated control of bacterial infections (including pneumonia). In addition, iNKT cells are essentially alloreactive, being restricted to a monomorphic CD1d molecule, allowing them to be used in an "off-the-shelf" and donor-independent manner (e.g., without causing graft-versus-host disease (GvHD)).

[0049] In some aspects, the present disclosure also provides a method for treating a subject suffering from acute respiratory distress syndrome (ARDS) (e.g., moderate or severe ARDS), the method comprising administering to a subject a composition comprising constant natural killer T (iNKT) cells. In certain embodiments, the present disclosure also provides a method for reducing or preventing organ failure in a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to a subject a composition comprising constant natural killer T (iNKT) cells. Acute respiratory distress syndrome (ARDS) and its milder forms of acute lung injury (ALI) are a series of lung diseases characterized by causing diffuse alveolar damage and leading to varying degrees of ventilation-perfusion mismatch, severe hypoxemia and a severe inflammatory process with poor lung compliance (Ware et al., The acute respiratory distress syndrome. N Engl J Med 2000; 342: 1334–49). ARDS is described as a rapid onset of shortness of breath and hypoxemia in otherwise healthy young individuals, accompanied by loss of lung compliance and bilateral infiltration on chest X-rays. Although the predisposing diseases of ARDS are different between patients, they have similar clinical and pathological features. The clinical syndrome associated with ARDS includes but is not limited to: (i) direct lung injury, such as lung infection (for example, viral or bacterial infection), pneumonia, aspiration of gastric contents, fat embolism, near drowning, inhalation injury, post-transplant reperfusion pulmonary edema and pulmonary embolectomy; and (ii) indirect lung injury, such as sepsis, trauma (for example, severe trauma with shock and multiple blood transfusions), cardiopulmonary bypass, transfusion of blood products and severe burns. In certain embodiments, the subject suffers from ARDS secondary to viral infection, and the viral infection includes but is not limited to coronavirus (for example, severe acute respiratory syndrome (SARS), SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV)), influenza (for example, H1N1, H5N1, H7N9), rhinovirus, herpes simplex virus (HSV), cytomegalovirus, parainfluenza virus, adenovirus, respiratory syncytial virus (RSV) or human metapneumovirus.

[0050] The Berlin definition defines ARDS patients into three mutually exclusive ARDS categories based on the degree of hypoxemia: mild (200 mm Hg < PaO2 / FIO2 ≤ 300 mm Hg), moderate (100 mm Hg < PaO2 / FIO2 ≤ 200 mm Hg), and severe (PaO2 / FIO2 ≤ 100 mm Hg). Using the Berlin definition, the stages of mild, moderate, and severe ARDS are associated with increased mortality (ARDS Definition Working Group et al., Acute Respiratory Distress Syndrome: the Berlin Definition, JAMA. 2012 Jun 20; 307(23): 2526-33).

[0051] In some embodiments, patients with ARDS are often mechanically ventilated during the course of their illness. Invasive mechanical ventilation (IMV) requires that the patient be intubated using an endotracheal tube (ETT) and a mechanical ventilator (as opposed to non-invasive ventilation where the interface is a mask). IMV helps stabilize patients with hypoxemia and hypercapnic respiratory failure, reduces the inspiratory work of breathing, redistributes blood flow from the working respiratory muscles to other tissues, and allows for lung protective (low tidal volume) ventilation. However, although IMV is an important care for patients in need (e.g., ARDS patients), mechanical ventilation itself can cause and further aggravate lung damage, and the mortality rate of patients undergoing IMV remains high (e.g., about 46%).

[0052] In some embodiments, the subject has ARDS secondary to SARS-CoV-2 infection. As used herein, "SARS-CoV-2" refers to the SARS-CoV having the nucleotide sequence of GenBank: MN996527.1 ("Severe acute respiratory syndrome coronavirus 2 isolate WIV02, complete genome"), reported in Zhou et al., Nature (2020) 579: 270–273, and encompasses SARS-CoV having at least 85% sequence identity with the nucleotide sequence of GenBank: MN996527.1. In some embodiments, the present invention relates to a variant of a nucleotide sequence having at least one of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequence identity. Variants of SARS-CoV-2 of particular interest include: (i) a variant designated VUI-202012 / 01, which belongs to the B.1.1.7 lineage and has a canonical nucleotide sequence of GISAID accession number EPI_ISL_601443; (ii) a variant designated 501Y.V2 / B.1.351, which has a canonical nucleotide sequence of GISAID accession number EPI_ISL_768642; (iii) a variant designated B.1.1.248 / P.1, which has a canonical nucleotide sequence of GISAID accession number EPI_ISL_792680; (iv) a variant designated B.1.617.1, which has a canonical nucleotide sequence of GISAID accession number EPI_ISL_2621960; and (v) a variant designated B.1.617.2, which has a canonical nucleotide sequence of GISAID accession number EPI_ISL_1663476. Variants of SARS-CoV-2 of particular interest also include variants designated α, β, γ, δ, δ+, κ, λ, μ, and o. The present disclosure relates to severe acute respiratory syndrome-related coronavirus (SARSr-CoV). The virology of SARSr-CoV and the epidemiology of diseases associated with SARSr-CoV infection are reviewed in, for example, Cheng et al., Clin Microbiol Rev (2007) 20(4): 660-694 and de Wit et al., Nat Rev Microbiol (2016) 14: 523–534.

[0053] In some embodiments, subjects with moderate ARDS as defined by the Berlin definition (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) receive iNKT cell therapy as described herein. In some embodiments, subjects with severe ARDS as defined by the Berlin definition (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) receive iNKT cell therapy as described herein. In some embodiments, subjects with ARDS (e.g., moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) receive IMV while receiving iNKT cell therapy. In some embodiments, iNKT cell therapy improves the survival rate of patients with ARDS (e.g., moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) relative to patients who do not receive iNKT cell therapy. In some embodiments, iNKT cell therapy improves the survival rate of patients with ARDS (e.g., moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) and placed in IMV, relative to patients who undergo IMV but do not receive iNKT cell therapy. In some embodiments, iNKT cell therapy achieves a 30-day survival rate (i.e., 30 days from initiation of IMV) of at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% in ARDS patients (e.g., patients with moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) undergoing IMV, compared to ARDS patients (e.g., patients with moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) who undergo IMV but do not receive iNKT cell therapy.

[0054] In some patients with severe ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) that is refractory to conventional therapy (e.g., when IMV is unable to maintain adequate oxygenation, and / or when IMV exacerbates lung injury), extracorporeal membrane oxygenation (ECMO) is used. In some embodiments, ARDS patients receive ECMO without previously receiving IMV. ECMO is a form of mechanical assist therapy that uses an extracorporeal blood circuit that includes an oxygenator and a pump. In order to perform standard respiratory ECMO, two vascular accesses are established, one for removing venous blood and the other for infusing oxygenated blood. Blood is drained from the main vein and pumped through a circuit that includes an oxygenator, which oxygenates the blood and removes carbon dioxide (CO2), after which the oxygenated blood is returned via another cannula. When the blood returns to the venous side of the circulation, the process is called veno-venous ECMO (VV ECMO), which provides gas exchange but cannot provide cardiac support. In some embodiments, patients with ARDS (e.g., patients with ARDS secondary to SARS-CoV-2 and / or influenza infection) are placed on VV ECMO while receiving iNKT cell therapy as described herein (e.g., a composition comprising iNKT cells). In some embodiments, VA ECMO is selected for patients with ARDS due to the need for cardiac support associated with pulmonary hypertension, cardiac dysfunction associated with sepsis, or arrhythmias.

[0055] In some embodiments, the present disclosure provides a method of treating a subject having ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) and undergoing ECMO (e.g., VV ECMO) using an iNKT cell therapy as described herein. In some embodiments, for example, iNKT cell therapy improves the survival of patients having ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) and undergoing ECMO (e.g., VV ECMO) compared to patients receiving ECMO alone. In some embodiments, iNKT cell therapy achieves a 90-day survival rate (i.e., 90 days from the start of ECMO) of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% in ARDS patients undergoing ECMO (e.g., patients with moderate or severe ARDS secondary to SARS-CoV-2 and / or influenza infection) compared to ARDS patients undergoing ECMO but not receiving iNKT cell therapy. In patients treated with iNKT cell therapy, no cell therapy-associated oxygenator failure due to filter clogging was observed, which is commonly seen in mesenchymal stem cell therapy for ARDS patients undergoing ECMO.

[0056] Due to the pro-inflammatory aspects of iNKT cells, the cytokine release syndrome (CRS) associated with cell therapy (e.g., CAR T therapy), and previous reports that iNKT cell activation exacerbates acute lung injury (see, e.g., Aoyagi et al., Activation of pulmonary invariant NKT cells leads to exacerbation of acute lung injury caused by LPS through local production of IFN-γand TNF-α by Gr-1+ monocytes, International Immunology, Vol. 23, No. 2, February 2011, pp. 97-108), the present disclosure also relates, in part, to the unexpected observation that iNKT cell therapy does not induce cytokine release syndrome (CRS) but promotes an anti-inflammatory response in ARDS patients (e.g., patients with ARDS secondary to SARS-CoV-2 and / or influenza infection). Cytokine release syndrome (CRS) is a systemic inflammatory response that results in the secretion of proinflammatory cytokines due to stimulation by a variety of factors, such as infection, immunotherapy (especially immunotherapy involving cell therapy), immune cell adaptors (e.g., T cell adaptors), and antibody-based therapies. In addition, severe respiratory infections (e.g., SARS-CoV-2 or influenza infection) are often associated with rapid viral replication, massive inflammatory cell infiltration, and elevated proinflammatory cytokine / chemokine responses, leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recent studies on experimentally infected animals have shown that virus-induced immunopathological events play a role in causing fatal pneumonia after infection. Cytokines are signaling molecules that can mediate and regulate the body's immune response and inflammation, which are protective under normal circumstances. However, when cytokine levels are too high, the immune response is overstimulated, healthy cells are destroyed, and vital organs are damaged. CRS can manifest itself in a variety of symptoms, ranging from flu-like symptoms to severe multi-organ system failure and even death. In some embodiments, subjects with ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) receiving iNKT cell therapy are monitored for the onset of CRS as measured by the production of proinflammatory cytokines. In some embodiments, the proinflammatory cytokines involved in CRS include, but are not limited to, IFN-γ, IL-1α / 1β, IL-5, IL-6, IL-7, IL-12, IL-17A, IP-10, TGFβ, CCL2, CCL5, CCL7, CXCL10, CXCL9, IL-8, ferritin, C-reactive protein (CRP), D-dimer, TNF-α, MCP01, or MIP-1α.Unexpectedly, iNKT cell therapy does not induce CRS in ARDS patients (e.g., patients with ARDS associated with SARS-CoV-2 and / or influenza infection). In some embodiments, IL-1α / β is not detected in ARDS patients receiving iNKT cell therapy, or is within the normal range for healthy subjects. In some embodiments, ferritin, CRP and / or D-dimer do not increase after iNKT cell therapy. In some embodiments, iNKT cell therapy promotes anti-inflammatory responses in ARDS patients (e.g., patients with ARDS associated with SARS-CoV-2 and / or influenza infection). In some embodiments, after administering iNKT cell therapy, serum levels of the anti-inflammatory cytokine IL-1RA (which counteracts IL-1-mediated cytokine release) in ARDS patients increase, indicating that iNKT cell therapy promotes anti-inflammatory responses.

[0057] Mortality in ARDS is often driven by multi-organ system failure (see, e.g., Siuba et al. Nonpulmonary Organ Failure in ARDS: What Can We Modify? Respiratory Care 2019 May, 64(5):610-611; Montgomery et al. Causes of mortality in patients with the adult respiratory distress syndrome. Am Rev Respir Dis 1985; 132(3):485–489; Stapleton et al. Causes and timing of death in patients with ARDS. Chest 2005; 128(2):525–532). This deterioration is thought to be secondary to extrapulmonary organ involvement due to a complex interaction between inflammatory mediators (e.g., CRS) and ongoing damage due to the ventilator mechanism. Common proinflammatory pathways due to initial injury (e.g., viral infection, sepsis, aspiration pneumonia, trauma) may exist between multiple organ system failure and ARDS (see, e.g., Han, The acute respiratory distress syndrome: from mechanism to translation. J Immunol 2015; 194(3): 855–860). Ventilator-associated lung injury has also been hypothesized to be a contributing factor to non-pulmonary organ failure (Slutsky AS et al., Multiple system organ failure. Is mechanical ventilation a contributing factor? Am J Respir Crit Care Med 1998; 157(6 Pt 1): 1721–1725; Tremblay et al., Ventilator-induced injury: from barotrauma to biotrauma. Proc Assoc Am Physicians 1998; 110(6): 482–488). In addition to respiratory failure, ARDS patients may develop dysfunction or failure of other organ systems, including but not limited to renal failure, liver failure, heart failure, blood system failure, and / or nervous system failure. In some embodiments, the present disclosure provides a method for reducing or preventing organ failure in a subject having acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.In some embodiments, iNKT cell therapy effectively prevents the occurrence of one or more organ failure or reduces the severity of one or more organ failure in ARDS patients receiving iNKT cell therapy relative to ARDS patients who have not received iNKT cell therapy. In some embodiments, iNKT cell therapy effectively prevents the occurrence of renal failure or reduces the severity of renal failure in ARDS patients receiving iNKT cell therapy relative to ARDS patients who have not received iNKT cell therapy. In some embodiments, iNKT cell therapy effectively reduces the number of ARDS patients with pulmonary organ failure relative to ARDS patients who have not received iNKT cell therapy.

[0058] ARDS patients are prone to concomitant infections (e.g., secondary lung infections, i.e., ventilator-associated pneumonia (VAP) or infections of other organs). The high frequency of VAP can be explained by traditional factors, such as bronchial contamination due to the duration of endotracheal intubation and mechanical ventilation (MV), but can also be explained by impaired local (alveolar) and systemic defenses and other specific and nonspecific factors (Papazian et al., Ventilator-associated pneumonia in adults: a narrative review. Intensive Care Med. 2020; Luty et al., Pulmonary infections complicating ARDS, Intensive Care Med. 2020; 46(12): 2168–2183). The incidence of concomitant infections includes, but is not limited to: pneumonia, bacteremia, urinary tract infection, fungemia, cytomegalovirus viremia, lung abscess, Klebsiella pneumonia, sepsis and septic shock or upper respiratory tract infection. The addition of venovenous extracorporeal membrane oxygenation (VV-ECMO) support has improved survival in patients with ARDS (eg, those with COVID-19), but overall mortality at 90 days remains low (ie, approximately 47%). Complications of VV-ECMO therapy include bleeding, oxygenator malfunction, and hospital-acquired infections, including but not limited to Klebsiella aerogenes, catheter-related bloodstream infection due to Candida albicans, and ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP) (see, e.g., Rivosecchi et al., Secondary Infections in Patients Requiring Extracorporeal Membrane Oxygenation (ECMO) for Severe Acute Respiratory Distress Syndrome (ARDS) due to COVID-19 Pneumonia (PNA), Open Forum Infectious Diseases, Vol. 8, No. 1 Suppl., November 2021, p. S260; Sun et al., Infections occurring during extracorporeal membrane oxygenation use in adult patients, The Journal of Thoracic and Cardiovascular Surgery, Vol. 140, No. 5, November 2010, pp. 1125-1132.e2).In some aspects, the present disclosure provides a method for reducing or preventing the concomitant infection of a subject with acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. In some aspects, the present disclosure also provides a method for reducing or preventing the concomitant infection of a subject with acute respiratory distress syndrome (ARDS) receiving invasive mechanical ventilation or veno-venous extracorporeal membrane oxygenation (VV ECMO), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. In certain embodiments, the concomitant infection of ARDS patients is a hospital-acquired infection, including but not limited to: Klebsiella aerogenes, catheter-related bloodstream infection due to Candida albicans, ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP). In certain embodiments, the hospital-acquired infection of ARDS patients causes pneumonia, bacteremia, urinary tract infection, fungemia, viremia (e.g., cytomegalovirus viremia), lung abscess, Klebsiella pneumonia, sepsis and septic shock or upper respiratory tract infection.

[0059] In some embodiments, the occurrence of concomitant infection in patients with ARDS (e.g., patients with ARDS secondary to SARS-CoV-2 and / or influenza infection) receiving iNKT cell therapy is monitored. In some embodiments, iNKT cell therapy reduces the occurrence of concomitant infection (e.g., hospital-acquired infection as described herein). In some embodiments, iNKT cell therapy prevents the occurrence of concomitant infection (e.g., hospital-acquired infection as described herein). In some embodiments, iNKT cell therapy is more effective at higher doses (e.g., at least 500 million cells, at least 600 million cells, at least 700 million cells, at least 800 million cells, at least 900 million cells, at least 1 billion cells or more) in preventing concomitant infection (e.g., hospital-acquired infection as described herein) than at lower doses (e.g., a dosage of less than 500 million cells) of iNKT cell therapy.

[0060] In certain embodiments, iNKT cells are administered after monitoring the lung function of ARDS patients (e.g., patients suffering from ARDS secondary to SARS-CoV-2 and / or influenza infection) receiving iNKT cell therapy. In certain embodiments, relative to the lung function of the subject before administration, the administration of iNKT cells causes the lung function of the subject to improve (e.g., lung function within 2 hours, within 5 hours, within 8 hours, within 12 hours, within 16 hours, within 24 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within two weeks, etc.). Pulmonary function can be measured by appropriate laboratory tests, e.g., lung capacity test, spirometry, X-ray, CT scan, etc. For example, in certain embodiments, relative to the lung capacity of the subject before administration, the administration of iNKT cells causes the lung capacity of the subject to increase (e.g., lung capacity within 2 hours, within 5 hours, within 8 hours, within 12 hours, within 16 hours, within 24 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within two weeks, etc.). In some embodiments, administration of iNKT cells results in an increase in lung parenchymal stability relative to the subject's lung parenchymal stability prior to administration (e.g., lung parenchymal stability is increased within 2 hours, within 5 hours, within 8 hours, within 12 hours, within 16 hours, within 24 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within one week, within two weeks, etc.).

[0061] Antibodies against exogenous HLA may be pathogenic in several clinical situations, most notably in transplantation (e.g., allogeneic cell therapy), where HLA antibodies may cause transplant rejection. For example, mesenchymal stem cell (MSC) therapy may cause humoral and cellular immune responses in the donor, particularly in allogeneic transplantation. Detection of donor-specific antibodies (DSA) in transplant recipient serum provides clear evidence that B cells recognize alloantigens. The generation of DSA is likely to be the result of indirect recognition of patient antigen-presenting cells (APCs) presenting donor HLA to CD4+ T cells. As a result, the induction of allogeneic specific T CD4+ cells will activate the B cells producing HLA-specific IgG (Barrachina et al., Allo-antibody productionafter intraarticular administration of mesenchymal stem cells (MSCs) in anequine osteoarthritis model: effect of repeated administration, MSCinflammatory stimulation, and equine leukocyte antigen (ELA) compatibility, StemCell Research & Therapy Vol. 11, Article No.: 52 (2020)). Other reports have shown that HLA antibodies are stable even after sustained CD19+ B cell depletion (e.g., Zhang et al., Stable HLA antibodies following sustained CD19+ cell depletion implicate a long-lived plasma cellsource, Blood Adv (2020) 4(18): 4292–4295). The long-term presence of DSA has a negative impact on the use of cell therapy. iNKT cell therapy can induce the production of DSA. In some embodiments, HLA matching reduces DSA induced by the allogeneic iNKT cell therapy described herein. In some embodiments, the incidence of DSA development by iNKT cell therapy decreases with increasing HLA class I matching. In some embodiments, contrary to previous reports, DSA induced by the iNKT cell therapy described herein is short-lived, thereby enabling the subject to be re-administered with the same iNKT cell therapy.

[0062] The iNKT cell therapy of the present disclosure (e.g., a composition comprising unmodified allogeneic iNKT cells) can be administered in a manner suitable for the disease to be treated or prevented (e.g., ARDS and its associated complications secondary to SARS-CoV-2 and / or influenza infection). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although the appropriate dose can be determined by clinical trials. In some embodiments, the compositions of the present disclosure are formulated for intravenous administration (e.g., intravenous injection or intravenous infusion).

[0063] When an "effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by the physician taking into account individual differences in age, weight, severity of ARDS, and condition of the patient (subject). In general, it can be said that the pharmaceutical composition comprising the iNKT cells described herein can be administered in an amount of 10 4 to 10 9 cells / kg body weight, 10 5 to 10 6 In some embodiments, iNKT cells may be administered at a dose of 80 million (i.e., 80 x 10 6 ) to 2 billion (i.e., 2000x10 6 ) cells (e.g., at least 80 million cells, at least 90 million cells, at least 100 million cells, at least 200 million cells, at least 300 million cells, at least 400 million cells, at least 500 million cells, at least 600 million cells, at least 700 million cells, at least 800 million cells, at least 900 million cells, at least 1 billion cells, at least 1.1 billion cells, at least 1.2 billion cells, at least 1.3 billion cells, at least 1.4 billion cells, at least 1.5 billion cells, at least 1.6 billion cells, at least 1.7 billion cells, at least 1.8 billion cells, at least 1.9 billion cells, at least 2 billion cells or more). iNKT cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0064] The administration of the subject composition can be carried out in any convenient manner, including infusion, injection, inhalation, ingestion, intratracheal injection, blood transfusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection or intraperitoneally. In certain embodiments, the iNKT cell composition of the present disclosure is administered to the patient by intradermal or subcutaneous injection. In certain embodiments, the iNKT cell composition of the present disclosure is preferably administered by iv injection or iv infusion. The composition of iNKT cells can also be directly injected into the disease site (e.g., intratracheal administration of ARDS patients undergoing IMV).

[0065] II. Unmodified allogeneic invariant natural killer T (iNKT) cells

[0066] In some aspects, the present disclosure provides a composition comprising invariant natural killer T (iNKT) cells. As used herein, the term "invariant natural killer T cell" or "invariant NKT cell," "iNKT cell," or "type I NKT cell" refers to a population of T lymphocytes expressing a conserved semi-constant TCR specific for lipid antigens restricted by the monomorphic MHC class I-related molecule CD1d. Natural killer T cells (NKT cells) were initially characterized in mice as T cells expressing both a TCR and NK1.1 (NKR-P1a-c or CD161), a C-type lectin NK receptor. Invariant NKT (iNKT) cells express a semi-constant αβ TCR (e.g., formed by the invariant TRAV11-TRAJ18 (4) rearrangement in mice, or by the cognate invariant TRAV10-TRAJ18 chain in humans), paired with a limited set of diverse Vβ chains, primarily TRBV1, TRBV29, or TRBV13 (6) in mice and TRBV25 in humans (e.g., see Dellabona et al., An invariant V alpha 24-J alpha Q / V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8-T cells. J Exp Med. (1994) 180: 1171–6.10.1084). Semi-constant TCRs recognize exogenous and endogenous lipid antigens presented by the monomorphic MHC class I-related molecule CD1d (see, e.g., Brennan et al., Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol. (2013) 13: 101-17.10.1038). Exogenous lipid antigens include the prototype α-galactosylceramide (α-GalCer) (Kawano et al., CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. (1997) 278: 1626-9.10.1126) and many bacterial-derived Ags, which can activate iNKT cells.

[0067] Compared with T cells, iNKT cells undergo different developmental pathways, resulting in the acquisition of innate effector functions in the thymus. Thymic iNKT cells do express markers that are usually upregulated by peripheral effector / memory T cells, such as CD44 and CD69, and unique NK differentiation markers, such as NK1.1 (in some mouse genetic backgrounds, CD161 in humans), CD122 (IL-2R / IL-15Rβ chain), CD94 / NKG2 and Ly49 (AJ), and a wide spectrum of TH1 / 2 / 17 effector cytokines. Once migrated to the periphery, iNKT cells form tissue-resident colonies that check cell integrity and quickly respond to local damage and inflammation, initiating reactions through cells of innate and adaptive immune responses.

[0068] Because iNKT cells can rapidly produce IFNγ, IL-4, or both, they have been found to play a role in various diseases by establishing an immune response based on either Th1 or Th2, depending on the context. In bacterial and viral infections, iNKT cells often help control pathogens early by establishing an effective Th1 response. In both mouse and human studies, the role of iNKT cells in diseases associated with excessive Th1 responses, such as type 1 diabetes and chronic obstructive pulmonary disease, has been described. The role of iNKT cells in helping to suppress Th1 responses and driving tolerogenic responses to transplants has also been described. As an example, after hematopoietic stem cell transplantation, the presence of iNKT cells can predict reduced survival in patients with graft-versus-host disease (GvHD) in both patients and preclinical models.

[0069] Therefore, the present disclosure provides a composition comprising iNKT cells. iNKT cell therapy can be autologous, allogeneic or xenogeneic. In some embodiments, iNKT cells are isolated from a donor (e.g., a donor that is not the subject). In some embodiments, iNKT cells are isolated from a subject. In some embodiments, iNKT cells are allogeneic. In some embodiments, the subject is a human and the donor is a human. In some embodiments, the subject and the donor are allogeneic. As used herein, the term "allogeneic" refers to a term for tissues and / or cells taken from different individuals of the same species, and the tissues and / or cells are genetically dissimilar and immunologically incompatible. iNKT cells are limited to single-state CD1d molecules and have essentially no alloreactivity, allowing them to be readily used in a donor-unrestricted manner (e.g., without causing graft-versus-host disease (GvHD)).

[0070] In some embodiments, iNKT cells are isolated (e.g., purified or enriched) from peripheral blood mononuclear cells (PBMCs) obtained from a single blood component of a donor. In some embodiments, the isolated iNKT cells are expanded in vitro. In some embodiments, the initial population of iNKT cells is purified from PBMCs using a suitable method known in the art (e.g., FACS or MACS). In certain embodiments, iNKT cells are isolated from PBMCs by microbead-bound monoclonal antibodies directed against the constant TCR of iNKTs.

[0071] In certain embodiments of the present invention, iNKT cells are stimulated in vitro. In certain embodiments, the initial population of iNKT cells is stimulated by α-galactosylceramide (α-GalCer) or any modified glycolipid thereof, for example, as described in Zhang et al., α-GalCer and iNKT Cell-Based Cancer Immunotherapy: Realizing the Therapeutic Potentials, Front Immunol. June 6, 2019; 10: 1126; Schafer et al., iNKT cell stimulation by glycolipid ligands modified from α-galactosylceramide resultsin differential interleukin-2secretion profiles, J Immunol May 1, 2019, 202 (1 supplement) 177.1), for activation and amplification. In certain embodiments, the initial population of iNKT cells is stimulated by α-GalCer when co-cultured with PBMC. In certain embodiments, PBMC is irradiated before co-culturing with iNKT cells. In some embodiments, PBMCs are pulsed with α-GalCer. In some embodiments, iNKT cells are stimulated with PBMCs pulsed with αGalCer. In some embodiments, iNKT cells can undergo more than one round of stimulation as described herein.

[0072] In certain embodiments, iNKT cells are expanded in vitro by culturing with IL-2, IL15, and / or IL-21. For example, in certain embodiments, iNKT cells are cultured in IL-2 for a period of time, wherein IL-2 is added to the culture medium once or multiple times. In certain embodiments, iNKT cells are expanded using IL-15 alone or together with IL-21. In certain embodiments, iNKT cells are expanded in vitro by culturing cells with IL-21. In certain embodiments, iNKT cells are expanded while being stimulated. In certain embodiments, iNKT cells are stimulated and then expanded. In certain embodiments, iNKT cells are expanded and then stimulated.

[0073] After expansion, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cells in the composition are iNKT cells.

[0074] In certain embodiments of the present invention, allogeneic iNKT cells are prepared, comprising the steps of: i) isolating iNKT cells from PBMCs by microbead-bound monoclonal antibodies against iNKT TCR, ii) stimulating PBMCs pulsed with the iNKT-specific ligand αGalCer, and iii) expanding iNKT cells driven by interleukin-2 (IL-2) for several weeks, followed by iv) cell culture harvesting, formulation, sterile filling, and cryopreservation.

[0075] In some embodiments, iNKT cells are unmodified (e.g., not genetically modified to express exogenous genes). The present disclosure also contemplates the use of iNKT cells isolated and / or expanded using any suitable method known in the art, for example, the methods described

[0076] In some embodiments, the expanded, unmodified iNKT cells express both Th1-type cytokines (e.g., IFNγ, TNFα, GM-CSF) and Th2-type cytokines (e.g., IL-4, IL-13). In some embodiments, after expansion, iNKT cells retain their intrinsic cytotoxic capacity against cells expressing CD1d. In some embodiments, the iNKT cell therapy composition is AgenT-797 (see, e.g., Yigit et al., 164AgenT-797, a novel allogeneic and "off-the-shelf" iNKT cell therapy promotes effective tumor killing.

[0077] The iNKT cells described herein can be characterized by reference to certain functional properties. In some embodiments, the iNKT cells described herein can have one or more of the following properties (e.g., when administered to a subject, such as a subject described herein and / or a subject treated as described herein): capable of treating SARS-CoV-2 (COVID-19); capable of treating moderate to severe SARS-CoV-2; capable of treating acute respiratory distress syndrome (ARDS); capable of treating moderate to severe ARDS; capable of treating moderate to severe ARDS secondary to SARS-CoV-2 or influenza; capable of treating patients with SARS-CoV-2 (and / or ARDS) who require invasive mechanical ventilation (IMV); capable of treating patients with SARS-CoV-2 (and / or ARDS) who require venovenous extracorporeal membrane oxygenation (VV ECMO); increasing survival after 30 days, e.g., for patients requiring IMV (e.g., at least 70% survival), compared to subjects not administered iNKT cells; increasing survival after 90 days, e.g., for patients undergoing VV ECMO, compared to subjects not administered iNKT cells; =The invention provides a novel method for the treatment of patients on ECMO (e.g., at least 70% survival rate); reduces the likelihood of oxygenator failure compared to subjects not administered with iNKT cells; reduces the risk / incidence of infection / development of concomitant infection (e.g., <40%) compared to subjects not administered with iNKT cells; reduces the risk / incidence of infection / development of pneumonia (e.g., ventilator-associated pneumonia (VAP)) compared to subjects not administered with iNKT cells; reduces the risk / incidence of organ failure of one or more organs compared to subjects not administered with iNKT cells; reduces the risk / incidence of renal failure, liver failure, hematological failure, and / or neurologic failure compared to subjects not administered with iNKT cells; does not induce cytokine release syndrome (CRS) associated with T cell therapy; induces an anti-inflammatory response; and reduces the risk / incidence of infection / development of pneumonia (e.g., ventilator-associated pneumonia (VAP)) compared to subjects not administered with iNKT cells. Compared with subjects not administered iNKT cells, the expression / secretion of proinflammatory cytokines (e.g., IL-2, IL-1α / 1β, IL-6, etc.) is reduced; compared with subjects not administered iNKT cells, the expression / secretion of anti-inflammatory cytokines (e.g., IL-1RA, etc.) is increased; the expression / secretion of growth factors (e.g., VEGF-D) is increased, anti-inflammatory response is increased; a favorable safety profile is exhibited (e.g., no adverse events are induced and / or adverse events occur during treatment); no dose-limiting toxicity is induced; a favorable cell persistence is exhibited (e.g., at least 28 days for a dose of 1 billion cells); translocation / distribution from blood to tissues is exhibited; a transient donor-specific alloantibody (DSA) response is induced; and / or the DSA response is reduced when the cells are HLA class I matched with the subject.

[0078] In some embodiments, the composition further comprises other components, such as cytokines (e.g., IL-2 or IL-15) or cell colonies. In short, the pharmaceutical composition of the present disclosure may include iNKT cell colonies as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0079] Pharmaceutical compositions of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although suitable dosage can be determined by clinical trials. In certain embodiments, compositions of the present disclosure are formulated for intravenous administration.

[0080] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description.Therefore, the specific embodiments should be interpreted as merely illustrative and not limiting of the remainder of the disclosure in any way.

[0081] Examples

[0082] The following examples are provided for illustrative purposes and are not intended to limit the scope of the present disclosure.

[0083] Example 1: Invariant Natural Killer T (iNKT) Cell Therapy in Subjects with Moderate Acute Respiratory Distress Syndrome Secondary to Viral Infection

[0084] The following examples show that iNKT cell therapy as described herein effectively improves survival; reduces inflammatory responses; reduces the occurrence or severity of concomitant infections (e.g., pneumonia); and / or reduces the occurrence or severity of post-ARDS organ failure in ARDS patients undergoing invasive mechanical ventilation or veno-venous extracorporeal membrane oxygenation (VV ECMO). In addition, iNKT cell therapy provides at least the following benefits: (i) exhibits a favorable safety profile (e.g., no neurotoxicity or grade ≥3 TRAEs were observed); (ii) exhibits short-term persistence in the periphery, consistent with in vivo data describing the rapid translocation of iNKT cells from blood to tissues; (iii) the opportunity for repeat dosing (although alloantibodies were detected after iNKT cell administration and were associated with the degree of HLA matching, the antibody response was short-lived); and (iv) the ability to treat viral diseases and infections (a reduced incidence of pneumonia was observed in patients receiving the highest dose of iNKT cell therapy).

[0085] Invariant natural killer T (iNKT) cells act as master regulators of immune responses, making them ideal for immunotherapy. This disclosure relates in part to the use of ex vivo expanded allogeneic iNKT cells to treat viral diseases of the lung, for the treatment of a broad spectrum of diseases, including viral diseases of the lung.

[0086] Without wishing to be bound by any particular theory, iNKT cells exert their antiviral effects via at least the following mechanisms: (i) direct viral killing: by recognizing CD1d ligands in diseased tissues and activating through the invariant TCR in a TCR-dependent manner; (ii) recruitment of host immunity by recruiting host T cells and NK cells: activating NK cells in a TCR-independent manner by recognizing stress signals through activating NK receptors (e.g., NKG2D, DNAM1); (iii) modulation and / or destruction of myeloid-derived suppressor cells and inflammatory monocytes that protect the airway epithelium; (iv) recruitment and activation of NK and T cells through cytokine secretion; (v) reversal of T cell exhaustion, such as restoration of the cytotoxic capacity, activation, and production of partially exhausted T cells; (vi) induction of maturation of immature dendritic cells; (vii) inhibition of proinflammatory cytokines; and (viii) cytokine-mediated control of bacterial infections, including pneumonia ( Figure 1 ).

[0087] A Phase 1 / 2 study is underway to evaluate the safety and potential efficacy of cell therapy using unmodified allogeneic invariant natural killer T (iNKT) cells in participants with moderate to severe acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 or influenza, who are either intubated or at high risk of intubation, as determined using the Berlin definition. Part 1 utilizes a standard 3+3 dose-escalation design of iNKT cells. All participants receive a single infusion of iNKT cells. Participants also receive other treatments and supportive care at the investigator's discretion. Once the maximum tolerated dose of iNKT cells is reached in Part 1, expansion cohorts will be opened. All participants are divided into three cohorts and receive a single infusion of varying doses of iNKT cells: Cohort 1 receives 100×10^6 iNKT cells; Cohort 2 receives 300×10^6 iNKT cells; and Cohort 3 receives 1000×10^6 iNKT cells.

[0088] Male and female subjects (aged >18 years) with evidence of SARS-Cov-2 infection and a diagnosis of moderate to severe ARDS secondary to SARS-Cov-2 or influenza according to the Berlin definition (2012).

[0089] The primary outcome measures included: (i) the number of patients who experienced adverse events during treatment; and (ii) the number of patients with dose-limiting toxicity (DLT).

[0090] Secondary outcomes included: (i) time to extubation (up to 30 days); (ii) mean daily Sequential Organ Failure Assessment score; (iii) change from baseline in C-reactive protein; (iv) decay in quantitative viral load from upper and lower respiratory tract samples; (v) time from dosing to viral clearance (up to day 30); and (vi) number of participants experiencing viral reactivation and fungal infection.

[0091] A summary of participant demographics by dose level cohort is shown in Table 1. Patients’ COVID-19 diagnosis dates were as follows: Figure 2 Patients were treated in a SARS-CoV-2 variant-agnostic manner at three research centers in the United States.

[0092] Table 1: Patient Demographics by Dose Level Cohort

[0093]

[0094]

[0095] After iNKT cell treatment, adverse reactions were evaluated, and representative results are shown in Table 2.

[0096] Table 2 Adverse reactions

[0097] Cohort 1 (n=3) Cohort 2 (n=4) Cohort 3 (n=13) Total (n=20) n(％) n(％) n(％) n(％) Any AE 3(100.0) 4(100.0) 13(100.0) 20(100.0) Any AE ≥ grade 3 3(100.0) 4(100.0) 12(92.3) 19(95.0) Any TRAE 1(33.3)2 3(75.0)5 1(7.7)1 5(25.0) Any TRAE ≥ grade 3 0 1(25.0) 0 1(5.0) Any TRAE leading to discontinuation 0 0 0 0 Any TRAE leading to dose interruption 0 0 0 0 Any TRAE resulting in death 0 0 0 0

[0098] Overall, iNKT cell therapy was well tolerated. Most observed adverse events (AEs) were consistent with the subject's underlying diagnosis of severe Covid-19 / ARDS requiring invasive mechanical ventilation (IMV). No dose-limiting toxicities were reported. Treatment-emergent adverse events (TEAEs) were observed in all subjects and were consistent with a diagnosis of severe COVID-19 / ARDS, including the most common anemia (n=8), fever (n=7), and acute kidney injury (n=6). One subject experienced a TRAE of ≥ grade 3 (dyspnea, grade 4).

[0099] Patient survival was analyzed by Kaplan-Mayer survival curves, which showed that the survival rate in the study group was 70% at 30 days after treatment, compared with 10% survival in the control group at 30 days after the start of IMV ( Figure 3 ).

[0100] In addition, the incidence of concomitant infections was analyzed for each cohort and is summarized in Table 3 .

[0101] Table 3. Cohort-level incidence of concomitant infections.

[0102]

[0103]

[0104] Cohort 3 (the highest dose level) showed a reduced incidence of concomitant infections. Compared to the incidence in cohorts 1 and 2, an approximate 50% reduction in the overall incidence of reported concomitant infections was observed in cohort 3 (100% in cohorts 1 and 2 compared to 46% in cohort 3). For the incidence of concomitant pneumonia cases, cohort 3 showed a reduction of more than 80% (15% incidence compared to 71%) compared to the combined number of cohorts 1+2. In comparison, the published incidence of ventilator-associated pneumonia (VAP) in COVID ARDS ranges from 25% to 84%.

[0105] Cytokine release syndrome (CRS) may be a feature of acute respiratory distress syndrome (ARDS) secondary to viral infection (e.g., influenza or SARS-Cov-2). Therefore, key indicators of CRS, such as serum levels of IL-1α, IL-1β, IL-6, ferritin, C-reactive protein (CRP), and D-dimer, were measured in subjects receiving iNKT cell therapy for 28 days. Cytokine data were grouped into pre-infusion samples (before, single time point), early post-infusion window (from 2 hours after infusion on day 1 to day 7 after infusion; D1-7) (which corresponds to the persistence of iNKT cells measured in the periphery), and late post-infusion time window (10th to 28th day; D10-28). Ferritin, CRP, D-dimer data were collected on the day of treatment (D1, single time point), early post-infusion window (2nd to 8th day; D2-8), and late post-infusion time window (8th to 28th day; D8-28). The dotted lines indicate the upper normal levels of the corresponding biomarkers in healthy individuals. Among the cytokines tested, IL-1α / β were not detected or within the normal range ( Figures 4A to 4B ); and IL-6, ferritin, CRP, and D-dimer were elevated in patients before infusion, and levels did not increase after infusion ( Figures 4C to 4F ). The results showed that treatment with iNKT cells did not induce CRS.

[0106] Patients with severe ARDS can undergo venovenous extracorporeal membrane oxygenation (VV ECMO), which may improve survival in these patients. However, mortality in ARDS patients undergoing VV ECMO remains high. In this study, patients treated with iNKT cells undergoing VV ECMO showed a 75% 90-day survival rate compared with control patients ( Figure 5). The median survival time for patients treated with iNKT cells (n=4) was 119.5 days, compared to 47 days in the control dataset (n=36). In ECMO, blood flows through tubes to artificial lungs in a machine that can add oxygen and remove carbon dioxide, allowing the patient's heart and lungs to rest. In patients treated with iNKT cells, no cell therapy-associated oxygenator failure due to filter clogging was observed, as has been seen with mesenchymal stem cell therapy in ARDS patients undergoing ECMO.

[0107] After iNKT cell infusion, the persistence of iNKT cells in the blood was assessed. Infused iNKT cells were quantified in patient PBMCs by digital PCR based on genetic markers unique to the donor material. Each line represents data from one patient. Infused iNKT cells were detected until day 6 after infusion, with possible low-level persistence being detectable at the highest dose level for longer periods, up to the last day of sampling at day 28 ( Figures 6A to 6C ). Peak levels of infused iNKT cells showed a dose proportional relationship. Data from patients undergoing ECMO are shown in red. Previous trials of mesenchymal stem cell therapy (MSC) in ECMO patients reported a high incidence of oxygenator failure due to filter clogging. The detection of infused iNKT cells long after infusion in ECMO patients is consistent with the absence of filter clogging in ECMO patients receiving iNKT cell therapy. Kinetics of tissue distribution of iNKT cells in a murine xenograft model showed rapid translocation of iNKT cells to tissues after intravenous injection ( Figure 6D The observed persistence of iNKT cells in patients' blood after brief infusion is consistent with the kinetics of blood-to-tissue distribution of iNKT cells in vivo.

[0108] Increased anti-inflammatory responses were observed after infusion of iNKT cell therapy ( Figures 7A to 7R ). Cytokines of particular interest are those known to play a key role in the pathogenesis of COVID-19, such as Figure 7A 、 7C , 7D, 7G, 7H, 7I, 7J, 7L, 7O and 7P. (The data of IL-1 and IL-6 are shown in Figures 4A to 4COf these IL-1RAs, serum levels showed the most significant changes after iNKT cell infusion, consistent with an increased anti-inflammatory response against IL-1-mediated cytokine release. Levels of the proinflammatory cytokine IL-7 were also significantly reduced. Data for IL-4, IL-15, IP-10, and VEGF-D were available only for cohorts 1 and 2. Significant changes after dosing are indicated by asterisks (*p<0.05; **p<0.01; data analyzed by one-way ANOVA). The dotted lines indicate the upper normal levels of the corresponding biomarkers in healthy individuals.

[0109] In addition, chest X-rays showed improved lung function in patients within 24 hours after iNKT cell infusion compared with chest X-rays before infusion. Figures 10A to 10B After iNKT cell infusion, the patient's lung capacity improved and parenchymal stabilization occurred.

[0110] The presence of donor-specific alloantibodies (DSA) was determined on the day of dosing and on day 14. Figures 8A to 8B Indicates the induction of DSA after iNKT cell infusion. Patients were scored for the induction of DSA on day 14 after infusion. The incidence of DSA development decreased with increasing HLA class I matching but did not appear to be related to the degree of HLA class II matching. Serum levels of DSA after administration decreased with increasing degree of HLA matching. DSA levels with a combined MFI < 1,000 were considered negative and were not reported ( Figure 8C For four patients, DSA levels were measured at discharge (days of patient discharge, from left to right: 60, 28, 21, 28). DSA levels after iNKT cell infusion peaked on day 14 and appeared to decline thereafter (data were normalized to peak DSA levels for each patient).

[0111] In summary, iNKT cell therapy represents a variant-agnostic approach for patients with ARDS (e.g., COVID-19 patients with ARDS). Patients treated with iNKT cells demonstrated a 70% on-study survival rate, compared to a 10% 30-day survival rate in the control group and a 39% CDC discharge outcome survival rate.

[0112] iNKT cell therapy demonstrated a favorable safety profile. No neurotoxicity or grade ≥3 TRAEs were observed. The MTD was not determined. iNKT cells also demonstrated transient persistence in the periphery, consistent with in vivo data describing rapid translocation of iNKT cells from blood to tissues. Alloantibody production correlated with the degree of HLA matching. However, antibody responses appeared to be transient, suggesting the possibility of re-dosing. In addition, a reduced incidence of pneumonia was observed in patients receiving the highest dose, highlighting the broader application of iNKT cells in viral diseases and infections.

[0113] Example 2: Safe administration of allogeneic iNKT cell infusions in patients with severe COVID-19 respiratory failure receiving venovenous extracorporeal membrane oxygenation (VV-ECMO) support

[0114] The following examples demonstrate that iNKT cell therapy as described herein is effective in improving survival; reducing pneumonia; and / or promoting anti-inflammatory responses in ARDS patients undergoing veno-venous extracorporeal membrane oxygenation (VV ECMO).

[0115] Although there has been a trend toward improvement in mortality in severe COVID-19 respiratory failure, primarily due to early corticosteroids and antiviral therapy, mortality remains high (47.9% to 84.4%). Adding venovenous extracorporeal membrane oxygenation (VV-ECMO) support has improved survival in a select group of COVID-19 patients, but the overall 90-day mortality remains at 47%. Complications of VV-ECMO therapy are numerous and include bleeding, oxygenator malfunction, and hospital-acquired infections. Multimodal therapies are needed to augment current interventions for patients with severe COVID-19 respiratory failure. Ideally, such therapies would modulate the initial excessive inflammatory response but could also enhance the innate immune system against other hospital-acquired threats. Invariant natural killer T (iNKT) cells comprise approximately 0.8% of leukocytes in healthy human hosts and are a natural home to damaged organs, including the lungs, where they suppress proinflammatory cytokines and protect epithelial tissues. Low circulating levels of iNKT cells may be a marker of COVID-19 ARDS mortality (Kreutmair et al., Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia, Immunity. 2021 Jul 13;54(7):1578-1593.e5).In severe cases of COVID-19, circulating iNKT cells have been shown to be activated by IL-18 (Jouan et al. (2020) Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients. J Exp Med 217(12)), a cytokine associated with unconventional T cell activation during viral infection (Tsai et al. Type I IFNs and IL-18 regulate the antiviral response of primary human γδT cells against dendritic cells infected with dengue virus. J. Immunol. 2015; 194(8): 3890–3900; Tyznik et al. Distinct requirements for activation of NKT and NK cells during viral infection. J. Immunol. 2014; 192(8): 3676-3685; Treiner E et al. (2003) Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422(6928):164–9. 10.1038 / nature01433). In addition, a decrease in circulating iNKT cells and IFNγ production by iNKT cells was observed.Among the remaining iNKTs in the circulation, levels of those expressing PD-1 and CD69 increased, and high PD-1 expression persisted on iNKT cells from patients in the intensive care unit on day 15 (Jouan et al., (2020) Phenotypical and functional alteration of unconventional T cells insevere COVID-19 patients. J Exp Med 217(12); Orumaa et al., The role of unconventional T cells in COVID-19, Ir J Med Sci. 2022; 191(2): 519–528; Liu et al., Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment, ASM Journals mBio, Vol. 12, No. 2).

[0116] In this study, four male patients with a mean age of 50 years with severe COVID-19 pneumonia met established criteria for venovenous extracorporeal membrane oxygenation (ECMO) cannulation. All four patients received dexamethasone and remdesivir and received a single 1000x 10 6 The primary endpoint was safety and tolerability, and multiple secondary and exploratory endpoints were identified, including all-cause mortality at 30 days.

[0117] No significant adverse infusion-related events, including oxygenator malfunction, were reported. Survival was 100% at 14 days and 75% at 30 and 90 days, with a mean ECMO run time of 133.5 days ( Figure 9A One patient died on day 15 from fulminant hepatic failure unrelated to study drug. The persistence of circulating iNKT cells in the VV-ECMO cohort was comparable to that in study patients not on ECMO ( Figure 9B ), and a reduced incidence of pneumonia was observed compared with the single-center VV-ECMO 2021-2022 cohort (n=38). Notably, the anti-inflammatory cytokine IL1-RA ( Figure 9C ) and the growth factor VEGF-D levels were significantly increased.

[0118] In patients with severe COVID-19 ARDS receiving venovenous extracorporeal membrane oxygenation (VV-ECMO), iNKT cell therapy was well tolerated. The overall 30-day survival rate was 75%. Exploratory data, such as hospital-acquired infection rates and protective cytokine levels in treated patients, showed statistically relevant and potentially important trends. These early observations support the efficacy of iNKT cell-based immunotherapy in treating ARDS, such as that associated with COVID-19.

[0119] Other embodiments

[0120] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0121] Based on the above description, those skilled in the art can easily determine the basic characteristics of the present disclosure, and without departing from the spirit and scope of the present disclosure, various changes and modifications can be made to the present disclosure to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0122] Equivalent

[0123] Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily devise various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, a person of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications in which the teachings of the present invention are used. Using only routine experimentation, a person of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced other than as specifically described and claimed. The present invention embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0124] It should be understood that all definitions, as defined and used herein, take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. Definitions of terms are disclosed throughout the specification, including but not limited to the "Definitions" section.

[0125] The section headings are not meant to be construed as limiting the scope of the disclosure.

[0126] Unless clearly indicated to the contrary, the indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one."

[0127] As used herein, the phrase "and / or" in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in conjunction in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to only A (optionally including elements other than B); in another embodiment, to only B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0128] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when listing items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of several elements or lists of elements, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of..." or "exactly one of..." or "consisting of..." when used in a claim will refer to the inclusion of exactly one element of multiple elements or lists of elements. Generally, when preceded by an exclusive term (such as "any one," "one of," "only one of," or "exactly one of"), the term "or" as used herein should only be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both"). When used in a claim, "consisting essentially of..." should have its ordinary meaning used in the field of patent law.

[0129] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting vector, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") may refer to at least one, optionally including more than one A and no B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one B and no A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one A, and to at least one, optionally including more than one B (and optionally including other elements); and so on.

[0130] It will also be understood that in any method claimed herein that includes more than one step or operation, the order of the steps or operations of the method is not necessarily limited to the recited steps or order of steps unless explicitly stated to the contrary.

[0131] In the claims and the foregoing specification, all transitional phrases such as "comprises," "includes," "carries," "has," "contains," "involves," "contains," "consisting of," etc. should be understood to be open-ended, meaning including but not limited to. As set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively. It should be understood that, in alternative embodiments, embodiments described in this document using open transitional phrases (e.g., "comprising") are also contemplated as "consisting of" and "consisting essentially of the features described by the open transitional phrases. For example, if the present disclosure describes a "composition comprising A and B," the present disclosure also contemplates the alternative embodiments "composition consisting of A and B" and "composition consisting essentially of A and B."

Claims

1. A method for treating a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

2. A method for reducing or preventing organ damage in a subject suffering from acute respiratory distress syndrome (ARDS) or at risk of organ failure, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

3. A method for inducing an anti-inflammatory response in a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

4. A method of reducing or preventing concomitant infection in a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

5. A method of reducing or preventing concomitant infection in a subject receiving invasive mechanical ventilation or venovenous extracorporeal membrane oxygenation (VV ECMO), the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

6. A method of reducing or preventing a hospital-acquired infection in a subject at risk of the infection, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT cells).

7. The method of claim 5 or claim 6, wherein the subject suffers from acute respiratory distress syndrome (ARDS).

8. The method according to any one of claims 1 to 7, wherein the iNKT cells are unmodified.

9. The method of any one of claims 1 to 8, wherein the iNKT cells are derived from a donor who is not the subject.

10. The method according to any one of claims 1 to 8, wherein the iNKT cells are allogeneic.

11. The method according to any one of claims 1 to 10, wherein the iNKT cells are isolated from peripheral blood mononuclear cells and expanded ex vivo.

12. The method according to any one of claims 1 to 11, wherein the donor is a human.

13. The method of any one of claims 1 to 12, wherein at least 90% of the cells in the composition are iNKT cells.

14. The method of any one of claims 1 to 13, wherein at least 95% of the cells in the composition are iNKT cells.

15. The method of any one of claims 1 to 14, wherein the ARDS is associated with a viral infection.

16. The method of claim 15, wherein the viral infection is caused by a coronavirus, influenza virus, rhinovirus, parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), or human metapneumovirus.

17. The method of claim 16, wherein the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-1), or Middle East respiratory syndrome coronavirus (MERS-CoV).

18. The method of claim 16, wherein the influenza virus is H1N1 influenza, H5N1 influenza, or H7N9 influenza.

19. The method of any one of claims 1 to 18, wherein the subject is not receiving mechanical ventilation.

20. The method of any one of claims 1 to 18, wherein the subject is receiving mechanical ventilation.

21. The method of claim 20, wherein the subject is mechanically ventilated while being administered the composition.

22. The method of any one of claims 1 to 21, wherein the subject is refractory to mechanical ventilation.

23. The method of any one of claims 1 to 22, wherein the subject is receiving extracorporeal membrane oxygenation (ECMO).

24. The method of claim 23, wherein the extracorporeal membrane oxygenation is venovenous extracorporeal membrane oxygenation (VVECMO).

25. The method of claim 24, wherein there is no oxygenator malfunction due to clogging.

26. The method of any one of claims 1 to 25, wherein administration of the composition does not induce cytokine release syndrome.

27. The method of any one of claims 1 to 26, wherein the administration of the composition improves the survival of the subject relative to a subject not administered the composition.

28. The method of any one of claims 1 to 27, wherein the administration of the composition induces an anti-inflammatory response in the subject as measured by one or more cytokines, wherein the one or more cytokines comprise: IL-1α / β, IL-6, ferritin, C-reactive protein (CRP), IL-2, IL-5, IL-7, IP-10, IL-15, IL-12p70, IFNγ, TFNα, IL-17A, IL-1RA, IL-4, IL-10, IL-13, IL-8, MCP-1, MIP-1α, VEGF or VEGF-D.

29. The method of any one of claims 1 to 28, wherein the administration of the composition reduces the occurrence of concomitant infection relative to a subject not administered the composition.

30. The method of any one of claims 4 to 5 and 7 to 29, wherein the concomitant infection is a hospital-acquired infection.

31. The method of claim 6 or claim 30, wherein the hospital-acquired infection comprises Klebsiella aerogenes, catheter-related bloodstream infection due to Candida albicans, ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP).

32. The method of any one of claims 1 to 31, wherein the administration of the composition reduces the occurrence of one or more organ failure relative to a subject not administered the composition.

33. The method according to any one of claims 1 to 32, wherein the organ failure comprises renal failure, liver failure, blood system failure and / or nervous system failure.

34. The method of any one of claims 1 to 32, wherein the organ failure is renal failure.

35. The method of any one of claims 1 to 34, wherein 80 x 10 6 Up to 2000x10 6 iNKT cells.

36. The method of any one of claims 1 to 35, wherein 100 x 10 6 iNKT cells.

37. The method of any one of claims 1 to 35, wherein 300 x 10 6 iNKT cells.

38. The method according to any one of claims 1 to 35, wherein 1000 x 10 6 iNKT cells.

39. The method of any one of claims 1 to 38, wherein the administration is via intravenous injection or intravenous infusion.

40. The method of any one of claims 1 to 39, wherein the composition is administered once to the subject.

41. The method of any one of claims 1 to 39, wherein the subject is capable of repeat dosing one or more times after an initial dosing.

42. The method of any one of claims 1 to 41, wherein the subject is also administered dexamethasone and / or remdesivir.

43. The method of any one of claims 1 to 42, wherein the administration results in an improvement in the subject's lung function compared to the subject's lung function before the administration.

44. The method of any one of claims 1 to 43, wherein the administration causes an increase in the lung capacity of the subject compared to the lung capacity of the subject before the administration.

45. The method of any one of claims 1 to 44, wherein the administering results in an increase in the stability of the subject's lung parenchyma compared to the stability of the subject's lung parenchyma before the administering.

46. ​​A method for reducing inflammation in a subject in need thereof, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

47. A method for reducing secondary infection in a subject in need thereof, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells.

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