Invariant 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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202380090195.0 (22) Application Date 2023.11.06 (30) Priority Data 63 / 423,036 2022.11.06 US 63 / 503,431 2023.05.19 US (85) PCT International Application Entering National Phase Date 2025.07.01 (86) PCT International Application Application Data PCT / US2023 / 078861 2023.11.06 (87) PCT International Application Publication Data WO2024 / 098075 EN 2024.05.10 (71) Applicant: MiNK Therapeutics, Inc. Address: New York, USA (72) Inventor: M. Purbuhu (74) Patent Agency: Beijing Yuhua United Intellectual Property Agency Co., Ltd. 11611 Patent Attorney: Liu Hualian (51) Int.Cl. A61K 35 / 17 (2006.01) A61P 11 / 00 (2006.01) A61P 31 / 00 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Constant Natural Killer T Cells for the Treatment of Acute Respiratory Distress Syndrome (ARDS) (57) Abstract: This disclosure relates at least in part to constant natural killer T cells containing constant natural killer T cells. Compositions of iNKT cells (e.g., unmodified allogeneic iNKT cells), and methods of using said composition comprising said 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). Claims 3 pages, Description 20 pages, Drawings 21 pages. CN 120826232 A 2025.10.21 CN 1 20 82 62 32 A 1. A method for treating a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising constant 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 constant 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 constant natural killer T (iNKT) cells.4. A method for reducing or preventing concomitant infections in a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. 5. A method for reducing or preventing concomitant infections in a subject receiving invasive mechanical ventilation or intravenous-venous extracorporeal membrane oxygenation (VV ECMO), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. 6. A method for reducing or preventing hospital-acquired infections in a subject at risk of such infections, the method comprising administering to the subject a composition comprising constant 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 of 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 not of the subject. 10. The method of any one of claims 1 to 8, wherein the iNKT cells are allogeneic. 11. The method of any one of claims 1 to 10, wherein the iNKT cells are isolated from peripheral blood mononuclear cells and expanded in vitro. 12. The method of any one of claims 1 to 11, wherein the donor is 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, H5N1, or H7N9 influenza. 19. The method of any one of claims 1 to 18, wherein the subject does not receive mechanical ventilation. 20. The method of any one of claims 1 to 18, wherein the subject receives mechanical ventilation. 21. The method of claim 20, wherein the subject receives mechanical ventilation while being administered the composition.Claims 1 / 3 Page 2 CN 120826232 A 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 receives extracorporeal membrane oxygenation (ECMO). 24. The method of claim 23, wherein the extracorporeal membrane oxygenation is venous-venous extracorporeal membrane oxygenation (VV ECMO). 25. The method of claim 24, wherein there is no oxygenator failure due to blockage. 26. The method of any one of claims 1 to 25, wherein the 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 rate of the subject compared to a subject who has not been administered the composition. 28. The method of any one of claims 1 to 27, wherein the administration of the composition to the subject induces an anti-inflammatory response, as measured by one or more cytokines, wherein the one or more cytokines include: 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 infections compared to subjects who have not been 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 includes *Klebsiella pneumoniae*, catheter-related bloodstream infection due to *Candida albicans*, and 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 incidence of one or more organ failures compared to a subject who has not been administered the composition. 33. The method of any one of claims 1 to 32, wherein the organ failure includes renal failure, liver failure, hematologic failure, and / or neurological 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 10⁶ to 2000 x 10⁶ iNKT cells are administered to the subject.36. The method of any one of claims 1 to 35, wherein 100 x 10⁶ iNKT cells are administered to the subject. 37. The method of any one of claims 1 to 35, wherein 300 x 10⁶ iNKT cells are administered to the subject. 38. The method of any one of claims 1 to 35, wherein 1000 x 10⁶ iNKT cells are administered to the subject. 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 to the subject once. 41. The method of any one of claims 1 to 39, wherein the subject is capable of repeat administration once or multiple times after the initial administration. 42. The method of any one of claims 1 to 41, wherein dexamethasone and / or remdesivir are also administered to the subject. 43. The method of any one of claims 1 to 42, wherein the administration causes an improvement in the lung function of the subject compared to the lung function prior to the administration. 44. The method of any one of claims 1 to 43, wherein the administration causes an increase in the lung volume of the subject compared to the lung volume of the subject prior to the administration. 45. The method of any one of claims 1 to 44, wherein the administration causes an increase in the stability of the lung parenchyma of the subject compared to the stability of the lung parenchyma prior to the administration. 46. A method for reducing inflammation in a subject with this need, the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. 47. A method for reducing secondary infection in a subject with this need, the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. Claims 3 / 3 Page 4 CN 120826232 A Constant Natural Killer T Cells for the Treatment of Acute Respiratory Distress Syndrome (ARDS)

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 423,036, filed November 6, 2022, and U.S. Provisional Application Serial No. 63 / 503,431, filed May 19, 2023, 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 to progressive dysfunction of other organs, also known as multiple system organ failure (MSOF).Effective treatments for reducing or preventing ARDS and post-ARDS MSOF remain elusive. Summary of the Invention

[0004] This disclosure relates at least in part to compositions comprising constant natural killer T (iNKT) cells (e.g., unmodified allogeneic iNKT cells), and methods of using compositions comprising these iNKT cells to treat a disease or its symptoms or complications (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 incidence or severity of pneumonia, and / or reduced incidence or severity of post-ARDS organ failure.

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

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

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

[0008] In some aspects, this disclosure provides 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 constant natural killer T (iNKT) cells.

[0009] In some aspects, this disclosure provides a method for reducing or preventing concomitant infections in a subject suffering from acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells.

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

[0011] In some embodiments, this disclosure provides a method for reducing or preventing hospital-acquired infections in subjects at risk of hospital-acquired infections, the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. In some embodiments, the subject at risk of hospital-acquired infection suffers from a viral infection. In some embodiments, the subject at risk of hospital-acquired infection is receiving invasive mechanical ventilation or venous-venous extracorporeal membrane oxygenation (VV ECMO). In some embodiments of the invention, the subject at risk of 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 in vitro.

[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 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-1 (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 (e.g., severe trauma with shock and multiple transfusions), cardiopulmonary bypass, transfusion of blood products, and severe burns.

[0018] In some embodiments, the subject does not receive mechanical ventilation.

[0019] In some embodiments, the subject receives mechanical ventilation 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 extracorporeal membrane oxygenation is venous-venous extracorporeal membrane oxygenation (VV ECMO). In some embodiments, there is no oxygenator failure 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 the survival rate of the subject compared to the subject who has not received 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 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 occurrence of concomitant infections (e.g., VAP) compared to subjects who have not received the composition. In some embodiments, the concomitant infection is a hospital-acquired infection. In some embodiments, hospital-acquired infections include Klebsiella pneumoniae, catheter-related bloodstream infections due to Candida albicans, and ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP).

[0026] In some embodiments, administration of the composition reduces the occurrence of one or more types of organ failure compared to subjects who have not received the composition. In some embodiments, organ failure includes renal failure, liver failure, hematologic failure, and / or nervous system failure. In some embodiments, organ failure is renal failure.

[0027] In some embodiments, 80 x 10⁶ to 2000 x 10⁶ iNKT cells are administered to the subject. In some embodiments, 100 x 10⁶ iNKT cells are administered to the subject. In some embodiments, 300 x 10⁶ iNKT cells are administered to the subject. In some embodiments, 1000 x 10⁶ iNKT cells are administered to the subject.

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

[0029] In some embodiments, the composition is administered to the subject once. In some embodiments, the subject may be able to repeat the administration once or more after the initial administration.

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

[0031] In some embodiments, administration causes improvement in the lung function of the subject compared to the lung function of the subject before administration. In some embodiments, administration causes an increase in the lung volume of the subject compared to the lung volume of the subject before administration. In some embodiments, administration causes an increase in the stability of the lung parenchyma of the subject compared to the stability of the lung parenchyma of the subject 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 1 is a schematic diagram illustrating the antiviral mechanism by constant natural killer T (iNKT) cells.

[0034] Figure 2 shows the date of COVID-19 diagnosis of patients in the context of the prevalence of SARS-CoV-2 strains in the United States.

[0035] Figure 3 shows the 30-day survival rate in the study compared to the survival outcome of the control group. Patients in the study showed a 70% survival rate compared to the 10% survival rate in the control group.

[0036] Figures 4A to 4F illustrate treatment with iNKT cell therapy for non-induced cytokine release syndrome (CRS). Figure 4A shows IL-1α levels before and after iNKT cell therapy. Figure 4B shows IL-1β levels before and after iNKT cell therapy. Figure 4C shows IL-6 levels before and after iNKT cell therapy. Figure 4D shows ferritin levels after iNKT cell therapy. Figure 4E shows C-reactive protein (CRP) levels after iNKT cell therapy. Figure 4F shows D-dimer levels after iNKT cell therapy.

[0037] Figure 5 shows the 90-day survival curves of patients undergoing VV ECMO with iNKT cell therapy compared to the control group (patients who underwent venous-venous extracorporeal membrane oxygenation (VV ECMO) but did not receive 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 show the peripheral persistence of iNKT cells in the blood of patients undergoing invasive mechanical ventilation (IMV) or VV ECMO. Figures 6A to 6C illustrate cohort-level peripheral persistence of iNKT cells in patient PBMCs via digital PCR based on donor-material-specific genetic markers. Each cohort received a different dose of iNKT cells. Each line represents data from one patient. Donor iNKT cells were detected up to day 6 post-infusion, with potentially lower levels of persistence at the highest dose levels lasting longer until the last day of sampling, day 28. Peak iNKT cell levels show dose-proportioning relationships. Data from patients undergoing ECMO are shown in red.Figures 6A to 6B are representative results from patients receiving iNKT cells in IMV. Figure 6C includes representative results from patients receiving iNKT cells in IMV or VV ECMO. Figure 6D illustrates the kinetics of tissue distribution of iNKT cells in a mouse xenograft model, showing rapid translocation of iNKT cells to tissues following intravenous (iv) injection. The observed persistence of iNKT cells after brief infusion in patient blood is consistent with the kinetics of blood-to-tissue distribution of iNKT cells in vivo.

[0039] Figures 7A to 7R are representative graphs showing serum cytokine levels of selected biomarkers across the immunomodulatory spectrum. Figures 7A to 7I are representative graphs showing the production of pro-inflammatory cytokines after iNKT cell infusion. Figures 7L to 7M show the production of anti-inflammatory cytokines after iNKT cell infusion. Figures 7N to 7P show the production of chemokines after iNKT cell infusion. Figures 7Q to 7R illustrate 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) identified on the day of administration and day 14. Figures 8A to 8B show the induction of DSA in patients who were HLA class I (Figure 8A) or HLA class II (Figure 8B). Figure 8C is a representative graph showing serum DSA levels after administration, which decreased with increasing HLA matching degree. DSA levels with MFI < 1,000 were considered negative and were not reported. Figure 8D is a representative graph showing DSA levels measured at discharge in four patients (patient discharge day, from left to right: 60, 28, 21, 28). DSA levels following iNKT cell infusion peaked on day 14 and appeared to decline thereafter (data normalized to peak DSA levels for each patient).

[0041] Figures 9A to 9C are representative graphs showing the survival rate, iNKT cell persistence, and anti-inflammatory cytokine production in patients undergoing venous-venous extracorporeal membrane oxygenation (VV-ECMO) and receiving iNKT cell therapy. Figure 9A shows a survival rate of 100% at 14 days and 75% at 30 and 90 days, with a mean ECMO duration of 133.5 days. Figure 9B shows that the persistence of circulating iNKT cells in the VV-ECMO cohort is comparable to that in study patients who did not undergo ECMO. Figure 9C shows a significant increase in the level of the anti-inflammatory cytokine IL-1-RA observed.

[0042] Figures 10A to 10B are chest X-ray images 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] This disclosure relates at least in part to compositions comprising constant natural killer T (iNKT) cells (e.g., unmodified allogeneic iNKT cells), and methods of using compositions comprising iNKT cells to treat diseases or symptoms or complications of diseases (e.g., acute respiratory distress syndrome (ARDS) secondary to a primary disease (e.g., viral infection)). In some embodiments, this disclosure is based on the following incidental observations: administration of iNKT cells to subjects (e.g., subjects with ARDS secondary to viral infection) resulted in improved survival rates in subjects receiving 1000 x 10⁶ iNKT cells (e.g., a 30-day survival rate of 70% for subjects undergoing IMV with iNKT cell therapy compared to a 10% 30-day survival rate for subjects undergoing invasive mechanical ventilation (IMV) but not receiving iNKT cell therapy; and a 90-day survival rate of 75% for subjects undergoing VV ECMO with iNKT cell therapy compared to approximately a 30% 90-day survival rate for subjects undergoing venous-venous extracorporeal membrane oxygenation (VV ECMO) but not receiving iNKT cell therapy), reduced inflammatory responses, reduced incidence or severity of concomitant infections and pneumonia, and / or reduced incidence or severity of post-ARDS organ failure. In addition, iNKT cell therapy provides at least the following benefits: (i) exhibiting a favorable safety profile (e.g., no neurotoxicity or ≥ grade 3 TRAE observed); (ii) exhibiting transient persistence in the periphery, consistent with in vivo data describing the rapid translocation of iNKT cells from blood to tissues; (iii) opportunities for repeated administration (although alloantibodies were detected after iNKT cell administration and correlated with 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] Additionally, this disclosure provides a variant-agnostic approach for ARDS (e.g., COVID-19 ARDS) and is the first immunocellular therapy used in patients undergoing ECMO. Although there has been a trend toward improved mortality in severe COVID-19 respiratory failure, primarily due to early corticosteroids and antiviral therapy, mortality remains high (47.9% to 84.4%). Increasing venous-venous extracorporeal membrane oxygenation (VV-ECMO) support improved survival rates in ARDS patients (e.g., ARDS in COVID-19 patients), but the overall mortality rate at 90 days remained low (i.e., approximately 47%).Complications of VV-ECMO therapy include bleeding, oxygenator failure, and hospital-acquired infections, including but not limited to Klebsiella pneumoniae, catheter-related bloodstream infections 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 therapies are required to enhance current interventions. This disclosure reports the first safe administration of allogeneic human unmodified constant natural killer T (iNKT) cell infusion in patients with severe COVID-19 respiratory failure receiving VV-ECMO support. No cell therapy-related oxygenator failure due to filter clogging was observed compared to previous mesenchymal stem cell therapy in ARDS patients undergoing ECMO.

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

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

[0047] In some aspects, this disclosure provides a method of treating a subject with a viral infection, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells. As used herein, the terms “administering” or “administration” mean providing a therapeutic agent (e.g., iNKT cells) or a composition thereof (e.g., a composition comprising iNKT cells) to a subject in a manner physiologically and / or pharmacologically useful (e.g., to treat a disease of the subject 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 rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a person who has or is suspected of having a disease. In some embodiments, the subject is a person who has or is suspected of having acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). As used herein, the terms “treating” or “treatment” mean applying or administering a composition comprising one or more active agents (e.g., unmodified allogeneic iNKT cells) to a subject suffering from a target disease or condition (e.g., ARDS), symptoms or complications of the disease / condition (e.g., respiratory distress, multiple organ failure), or susceptibility or primary indication of the disease / condition (e.g., viral infection) with the aim of curing, resolving, alleviating, altering, remedying, improving, ameliorating, or influencing the condition (e.g., ARDS), symptoms or complications of the disease (e.g., respiratory distress, multiple organ failure), or susceptibility or primary indication of the disease / condition (e.g., viral infection).Alleviating the target disease / symptom includes delaying or preventing the development or progression of the disease, reducing the severity of the disease, and / or promoting survival.

[0048] iNKT cell therapy elicits antiviral effects at least in the following ways: (i) recognizing CD1d ligands in diseased tissues and activating them via a constant TCR; (ii) recognizing stress signals by activating NK receptors (e.g., NKG2D, DNAM1); (iii) modulating and / or disrupting myeloid suppressor cells and inflammatory monocytes; (iv) recruiting and activating NK cells and T cells through cytokine secretion; (v) reversing T cell exhaustion; and (vi) controlling cytokine-mediated bacterial infections (including pneumonia). Furthermore, iNKT cells are essentially non-allogeneic and limited to singlet CD1d molecules, allowing them to be used in a “ready-to-use” and donor-unrestricted manner (e.g., without causing graft-versus-host disease (GvHD)).

[0049] In some aspects, this disclosure also provides a method for treating 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 some embodiments, this 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 the subject a composition comprising constant natural killer T (iNKT) cells. Acute respiratory distress syndrome (ARDS) and its milder form, acute lung injury (ALI), are a group of lung diseases characterized by a severe inflammatory process that causes diffuse alveolar damage and results in varying degrees of ventilation-perfusion mismatch, severe hypoxemia, and 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 tachypnea and hypoxemia in otherwise healthy young individuals, accompanied by loss of lung compliance and bilateral infiltrates on chest X-ray. Although the precipitating diseases of ARDS vary among patients, they share similar clinical and pathological features. Clinical syndromes associated with ARDS include, but are not limited to: (i) direct lung injury, such as lung infections (e.g., viral or bacterial infections), pneumonia, aspiration of gastric contents, fat embolism, near drowning, aspiration injury, post-transplant reperfusion pulmonary edema, and pulmonary embolism resection; and (ii) indirect lung injury, such as sepsis, trauma (e.g., severe trauma with shock and multiple transfusions), cardiopulmonary bypass, transfusion of blood products, and severe burns.In some embodiments, the subject suffers from ARDS secondary to a viral infection, including but not limited to coronaviruses (e.g., severe acute respiratory syndrome (SARS), SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV)), influenza (e.g., 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 based on the degree of hypoxemia into three mutually exclusive ARDS categories: 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, staging of mild, moderate, and severe ARDS is associated with increased mortality (ARDS Definition Working Group et al., Acute Respiratory Distress Syndrome: the Berlin Definition, JAMA. 20 June 2012; 307(23):2526-33).

[0051] In some embodiments, patients with ARDS are frequently mechanically ventilated during their illness. Invasive mechanical ventilation (IMV) requires intubation using an endotracheal tube (ETT) and a mechanical ventilator (as opposed to noninvasive ventilation with a face mask). IMV helps stabilize patients with hypoxemia and hypercapnia-related respiratory failure, reduces the work of breathing, redistributes blood flow from the moving respiratory muscles to other tissues, and allows for lung-protective (low tidal volume) ventilation. However, while IMV is an important form of care for patients in need (e.g., patients with ARDS), mechanical ventilation itself can cause and further exacerbate lung injury, and mortality rates for patients undergoing IMV remain high (e.g., approximately 46%).

[0052] In some embodiments, the subject had ARDS secondary to SARS-CoV-2 infection.As used herein, “SARS-CoV-2” refers to SARS-CoV having the nucleotide sequence of GenBank:MN996527.1 (“Severe Acute Respiratory Syndrome Coronavirus 2 Isolator WIV02, Complete Genome”), reported in Zhou et al., Nature (2020) 579:270–273, and encompasses those having at least 85% sequence identity with the nucleotide sequence of GenBank:MN996527.1 (e.g., at least 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%). A variant of the nucleotide sequence (or one of the higher sequence identity). Variants of particular interest in SARS-CoV-2 include: (i) a variant named VUI-202012 / 01 belonging to the B.1.1.7 lineage with a canonical nucleotide sequence of GISAID accession number EPI_ISL_601443; (ii) a variant named 501Y.V2 / B.1.351 with a canonical nucleotide sequence of GISAID accession number EPI_ISL_768642; (iii) a variant called B.1.1.248 / P.1 with a canonical nucleotide sequence of GISAID accession number EPI_ISL_792680; (iv) a variant called B.1.617.1 with a canonical nucleotide sequence of GISAID accession number EPI_ISL_2621960; and (v) a variant called B.1.617.2 with a canonical nucleotide sequence of GISAID accession number EPI_ISL_1663476. Variants of particular interest in SARS-CoV-2 also include those called α, β, γ, δ, δ+, κ, λ, μ, and ο. This disclosure relates to severe acute respiratory syndrome-associated coronavirus (SARSr-CoV). A virological review of SARSr-CoV and an epidemiological review of diseases associated with SARSr-CoV infection can be found, for example, in Cheng et al., Clin Microbiol Rev (2007) 20(4):660-694 and de Wit et al., Nat Rev Microbiol (2016) 14:523–534. Specification 6 / 20 pages 10 CN 120826232 A

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

[0054] Extracorporeal membrane oxygenation (ECMO) is used in some patients with severe refractory ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) that respond to conventional therapy (e.g., when IMV fails to maintain adequate oxygenation, and / or when IMV exacerbates lung injury). In some embodiments, ARDS patients receive ECMO without prior IMV treatment. ECMO is a form of mechanically assisted therapy that employs an extracorporeal blood circuit including an oxygenator and a pump. For 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 including an oxygenator that oxygenates the blood and removes carbon dioxide (CO2), after which the oxygenated blood is returned via another cannula. This process, where blood is returned to the venous side of the circulation, is called venous-venous ECMO (VV ECMO), which provides gas exchange but not cardiac support. In some embodiments, ARDS patients (e.g., those with ARDS secondary to SARS-CoV-2 and / or influenza infection) undergo VV ECMO while receiving the iNKT cell therapy described herein (e.g., a composition containing iNKT cells).In some embodiments, VA ECMO is selected for ARDS patients due to the need for cardiac support associated with pulmonary hypertension, cardiac dysfunction associated with sepsis, or arrhythmias.

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

[0056] Due to the pro-inflammatory aspects of iNKT cells, cytokine release syndrome (CRS) associated with cell therapy (e.g., CAR T therapy), and previous reports on iNKT cell activation exacerbating acute lung injury (see, for example, Aoyagi et al., Activation of pulmonary invariant NKT cells leads to exacerbation of acute lung injury caused by LPS through local production of IFN-γ and TNF-α by Gr-1+, specification 7 / 20 pages 11 CN 120826232 A monocytes, International Immunology, Vol. 23, No. 2, February 2011, pp. 97-108), this disclosure also relates in part to the following unexpected observation: iNKT cell therapy does not induce cytokine release syndrome (CRS), but promotes anti-inflammatory responses in patients with ARDS (e.g., patients with ARDS secondary to SARS-CoV-2 and / or influenza infection). Cytokine release syndrome (CRS) is a systemic inflammatory response resulting from the excessive secretion of pro-inflammatory cytokines due to stimulation by various factors, such as infection, immunotherapy (especially cell-based immunotherapy), immune cell adjuvants (e.g., T-cell adjuvants), and antibody-based therapies. Furthermore, severe respiratory infections (e.g., SARS-CoV-2 or influenza infection) are often associated with rapid viral replication, extensive inflammatory cell infiltration, and elevated pro-inflammatory cytokine / chemokine responses, leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recent studies in experimentally infected animals have shown that virus-induced immunopathological events play a role in causing fatal pneumonia after infection. Cytokines are signaling molecules that mediate and regulate the body's immune response and inflammation, and they are protective under normal conditions. However, when cytokine levels are excessively high, they overstimulate the immune response, destroying healthy cells and damaging vital organs. CRS can manifest in a variety of symptoms, ranging from flu-like symptoms to severe multi-organ failure and even death. In some embodiments, the onset of CRS in subjects with ARDS (e.g., ARDS secondary to SARS-CoV-2 and / or influenza infection) who receive iNKT cell therapy is monitored, such as by measurement of the production of pro-inflammatory cytokines.In some embodiments, pro-inflammatory 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. Surprisingly, 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 as in 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 an anti-inflammatory response in ARDS patients (e.g., patients with ARDS associated with SARS-CoV-2 and / or influenza infection). In some embodiments, an increase in serum levels of the anti-inflammatory cytokine IL-1RA (which counteracts IL-1-mediated cytokine release) in ARDS patients after administration of iNKT cell therapy indicates that iNKT cell therapy promotes an anti-inflammatory response.

[0057] Mortality in ARDS is often driven by multiple organ system failure (see, for example, Siuba et al., Nonpulmonary Organ Failure in ARDS: What Can We Modify? Respiratory Care May 2019, 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, resulting from a complex interaction between inflammatory mediators (e.g., CRS) and persistent damage caused by the ventilator mechanics.Common pro-inflammatory pathways arising from initial injury (e.g., viral infection, sepsis, aspiration pneumonia, trauma) may exist between multiple organ system failure and ARDS (see, for example, Han, The acute respiratory distress syndrome: from mechanism to translation. J Immunol 2015; 194(3):855–860). Ventilator-associated lung injury has also been presumed 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(6Pt 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, patients with ARDS may develop dysfunction or failure of other organ systems, including but not limited to renal failure, hepatic failure, heart failure, hematologic failure, and / or neurological failure. In some embodiments, this disclosure provides a method for reducing or preventing organ failure in a subject suffering from acute respiratory distress syndrome (ARDS), comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. In some embodiments, iNKT cell therapy effectively prevents the occurrence of one or more organ failures or reduces the severity of one or more organ failures 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 comorbid infections (e.g., secondary pulmonary infections, i.e., ventilator-associated pneumonia (VAP) or infections of other organs).The high frequency of VAP can be explained by conventional factors, such as bronchial contamination due to the duration of endotracheal intubation and mechanical ventilation (MV), but can also be explained by compromised local (alveolar) and systemic defenses, as well as 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 comorbid infections includes, but is not limited to: pneumonia, bacteremia, urinary tract infection, fungemia, cytomegalovirus viremia, lung abscess, Klebsiella pneumoniae, sepsis and septic shock, or upper respiratory tract infection. Increased venous-venous extracorporeal membrane oxygenation (VV-ECMO) support has improved survival in patients with ARDS (e.g., ARDS in patients with COVID-19), but the overall mortality rate at 90 days remains low (i.e., approximately 47%). Complications of VV-ECMO therapy include bleeding, oxygenator failure, and hospital-acquired infections, including but not limited to Klebsiella pneumoniae, catheter-related bloodstream infections due to Candida albicans, and ventilation-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (see, for example, 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, Supplement 1, 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, this disclosure provides a method for reducing or preventing concomitant infections in subjects 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, this disclosure also provides a method for reducing or preventing concomitant infections in subjects with ARDS receiving invasive mechanical ventilation or intravenous-venous extracorporeal membrane oxygenation (VV ECMO), the method comprising administering to the subject a composition comprising constant natural killer T (iNKT) cells. In some embodiments, the concomitant infection in ARDS patients is a hospital-acquired infection, including but not limited to: Klebsiella pneumoniae, catheter-related bloodstream infections due to Candida albicans, and ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP). In some embodiments, hospital-acquired infections in ARDS patients result in pneumonia, bacteremia, urinary tract infections, fungemia, viremia (e.g., cytomegalovirus viremia), lung abscess, Klebsiella pneumoniae, sepsis and septic shock, or upper respiratory tract infections.

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

[0060] In some embodiments, lung function in ARDS patients receiving iNKT cell therapy (e.g., patients with ARDS secondary to SARS-CoV-2 and / or influenza infection) is monitored after iNKT cell administration. In some embodiments, iNKT cell administration results in an improvement in the subject's lung function relative to the subject's lung function before administration (e.g., improvement in lung function within 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, etc.). Lung function can be measured by appropriate laboratory tests, such as lung volume testing, vital capacity measurement, X-ray, CT scan, etc.For example, in some embodiments, administration of iNKT cells resulted in an increase in the lung volume of the subject relative to the lung volume prior to administration (e.g., an increase in lung volume within 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, etc.). In some embodiments, administration of iNKT cells resulted in an increase in lung parenchymal stability relative to the lung parenchymal stability of the subject prior to administration (e.g., an increase in lung parenchymal stability within 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, etc.).

[0061] Antibodies against exogenous HLA can be pathogenic in several clinical situations, most notably in transplantation (e.g., allogeneic cell therapy), where HLA antibodies can lead to transplant rejection. For example, mesenchymal stem cell (MSC) therapy can elicit humoral and cellular immune responses in the donor, particularly in allogeneic transplantation. The detection of donor-specific antibodies (DSA) in transplant recipient serum provides clear evidence of B cell recognition of allogeneic antigens. DSA production is likely a result of indirect recognition of donor HLA by patient antigen-presenting cells (APCs) presenting it to CD4+ T cells. Consequently, induction of allogeneic specific T CD4+ cells activates B cells that produce HLA-specific IgG (Barrachina et al., Allo-antibody production after intraarticular administration of mesenchymal stem cells (MSCs) in an equine osteoarthritis model: effect of repeated administration, MSC inflammatory stimulation, and equine leukocyte antigen (ELA) compatibility, Stem Cell Research & Therapy, Vol. 11, 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 cell source, Blood Adv (2020) 4(18): 4292–4295).The long-term presence of DSA negatively impacts the use of cell therapy. iNKT cell therapy can induce 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 transient, thereby enabling re-dosing of the subject with the same iNKT cell therapy.

[0062] The iNKT cell therapy of this 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 through clinical trials. In some embodiments, the compositions of this disclosure are formulated for intravenous administration (e.g., intravenous injection or intravenous infusion).

[0063] When referring to an “effective amount” or a “therapeutic amount,” the precise amount of the composition of this disclosure to be administered can be determined by a physician taking into account individual differences in the patient (subject)’s age, weight, severity of ARDS, and condition. Generally speaking, a pharmaceutical composition containing iNKT cells described herein can be administered at a dose of 10⁴ to 10⁹ cells / kg body weight, 10⁵ to 10⁶ cells / kg body weight (inclusive of all integer values ​​within those ranges). In some embodiments, iNKT cells may be administered in doses ranging from 80 million (i.e., 80 x 10⁶) to 2 billion (i.e., 2000 x 10⁶) 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). The iNKT cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

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

[0065] II. Unmodified Allogeneic Invariant Natural Killer T (iNKT) Cells

[0066] In some aspects, this disclosure provides a composition comprising invariant natural killer T (iNKT) cells. As used herein, the terms “constant natural killer T cells” or “constant NKT cells,” “iNKT cells” or “type I NKT cells” refer to a population of T lymphocytes that express a conserved semi-constant TCR specific to a lipid antigen restricted by singlet MHC class I-associated molecule CD1d. Natural killer T cells (NKT cells) were initially characterized in mice as T cells expressing both TCR and NK1.1 (NKR-P1a-c or CD161) (a type C lectin NK receptor). Constant NKT (iNKT) cells express semi-constant αβTCR (e.g., formed by constant TRAV11-TRAJ18(4) rearrangement in mice, or by homologous constant TRAV10-TRAJ18 chain in humans), which pair 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 11T 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 singlet MHC class I-related molecule CD1d (see, for example, Brennan et al., Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat RevImmunol. (2013)13:101-17.10.1038). Exogenous lipid antigens include proto-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 Ag, which can activate iNKT cells.

[0067] Compared with T cells, iNKT cells undergo a different developmental pathway, resulting in innate effector functions already acquired in the thymus. Thymic iNKT cells do express markers typically upregulated by peripheral effector / memory T cells, such as CD44 and CD69, as well as unique NK differentiation markers, such as NK1.1 (CD161 in some mouse genetic backgrounds and in humans), CD122 (IL-2R / IL-15Rβ chains), CD94 / NKG2 and Ly49 (A-J), and a broad spectrum of TH1 / 2 / 17 effector cytokines. Once migrated to the periphery, iNKT cells form a tissue-resident population that examines cellular integrity and responds rapidly to local injury and inflammation, initiating responses through 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 environment-dependent Th1 or Th2-based immune responses. In bacterial and viral infections, iNKT cells often help control pathogens early by establishing effective Th1 responses. In two studies in mice and humans, the role of iNKT cells in diseases such as type 1 diabetes and chronic obstructive pulmonary disease associated with excessive Th1 responses, as described on page 11 / 20 of the specification (CN 120826232 A). The role of iNKT cells in helping to suppress Th1 responses and drive tolerance-inducing responses to grafts has also been described. As an example, the presence of iNKT cells after hematopoietic stem cell transplantation can predict reduced survival rates of graft-versus-host disease (GvHD) in patients and preclinical models.

[0069] Therefore, this 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., not a recipient). In some embodiments, iNKT cells are isolated from a recipient. In some embodiments, iNKT cells are allogeneic. In some embodiments, the recipient is human and the donor is human. In some embodiments, the recipient and the donor are allogeneic. As used herein, the term “allogeneic” refers to 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 confined to singlet CD1d molecules and are substantially non-allogeneic, allowing them to be used off-the-shelf 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) derived from apheresis blood components from a donor. In some embodiments, the isolated iNKT cells are expanded in vitro. In some embodiments, an initial population of iNKT cells is purified from PBMCs using suitable methods known in the art (e.g., FACS or MACS). In some embodiments, iNKT cells are isolated from PBMCs by microbead-bound monoclonal antibodies targeting the iNKT constant TCR.

[0071] In some embodiments of the invention, iNKT cells are stimulated in vitro. In some embodiments, the initial population of iNKT cells is stimulated with 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. 2019, 6 June; 10:1126; Schafer et al., iNKT cell stimulation by glycolipid ligands modified from α-galactosylceramide results in differential interleukin-2 secretion profiles, J Immunol. 2019, 1 May; 202(1 Supplement) 177.1), for activation and expansion. In some embodiments, the initial population of iNKT cells is stimulated with α-GalCer while co-cultured with PBMCs. In some embodiments, the PBMCs are irradiated prior to co-culturing with iNKT cells. In some embodiments, the PBMCs are pulsed with α-GalCer.In some embodiments, PBMCs irradiated with αGalCer pulses are used to stimulate iNKT cells. In some embodiments, iNKT cells may undergo more than one round of stimulation as described herein.

[0072] In some embodiments, iNKT cells are expanded in vitro by culturing with IL-2, IL-15, and / or IL-21. For example, in some 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 some embodiments, iNKT cells are expanded with IL-15 alone or together with IL-21. In some embodiments, iNKT cells are expanded in vitro by culturing cells with IL-21. In some embodiments, iNKT cells are expanded simultaneously with stimulation. In some embodiments, iNKT cells are stimulated and then expanded. In some 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 some embodiments of the present invention, the preparation of allogeneic iNKT cells includes the following steps: i) isolating iNKT cells from PBMCs using a microbead-bound monoclonal antibody against iNKT TCR, ii) stimulating PBMCs with pulsed irradiation of the iNKT-specific ligand αGalCer, and iii) expanding iNKT cells driven by interleukin-2 (IL-2) over several weeks, followed by iv) harvesting, preparing, aseptically filling, and cryopreserving the cell culture.

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

[0076] In some embodiments, the expanded, unmodified iNKT cells express both Th1 cytokines (e.g., IFNγ, TNFα, GM-CSF) and Th2 cytokines (e.g., IL-4, IL-13). In some embodiments, after expansion, the iNKT cells retain their inherent cytotoxicity against cells expressing CD1d. In some embodiments, the iNKT cell therapy composition is AgenT-797 (see, for example, Yigit et al., 164). AgenT-797 is a novel allogeneic and “off-the-shelf” iNKT cell therapy that 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 may have one or more of the following characteristics (e.g., when administered to subjects, such as those described herein and / or those 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) requiring invasive mechanical ventilation (IMV); capable of treating patients with SARS-CoV-2 (and / or ARDS) requiring intravenous-venous extracorporeal membrane oxygenation (VV ECMO); increased survival rate after 30 days compared to subjects not administered iNKT cells, e.g., for patients requiring IMV (e.g., at least 70% survival); increased survival rate after 90 days compared to subjects not administered iNKT cells, e.g., for patients undergoing VV ECMO. Patients on ECMO (e.g., at least 70% survival); reduced likelihood of oxygenator failure compared to subjects not receiving iNKT cells; reduced risk / incidence of infection / development of concomitant infections (e.g., <40%) compared to subjects not receiving iNKT cells; reduced risk / incidence of infection / development of pneumonia (e.g., ventilator-associated pneumonia (VAP)) compared to subjects not receiving iNKT cells; reduced risk / incidence of organ failure of one or more organs compared to subjects not receiving iNKT cells; reduced risk / incidence of renal failure, hepatic failure, hematologic failure, and / or neurological failure compared to subjects not receiving iNKT cells; did not induce T-cell therapy-related cytokine release syndrome (CRS); induced anti-inflammatory response; increased anti-inflammatory response compared to subjects not receiving iNKT cells; reduced expression / secretion of pro-inflammatory cytokines (e.g., IL-2, IL-1α / 1β, IL-6, etc.) compared to subjects not receiving iNKT cells; increased expression / secretion of anti-inflammatory cytokines (e.g., IL-1RA, etc.) compared to subjects not receiving iNKT cells; Increases the expression / secretion of growth factors (e.g., VEGF-D), anti-inflammatory response; exhibits favorable safety profile (e.g., does not induce adverse events and / or adverse events occurring during treatment); does not induce dose-limiting toxicity; exhibits favorable cell persistence (e.g., at least 28 days for a dose of 1 billion cells); exhibits translocation / distribution from blood to tissues; induces transient donor-specific alloantibody (DSA) response; and / or reduces DSA response when cells are matched with the subject's HLA class.

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

[0079] The pharmaceutical compositions of this disclosure may 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 an appropriate dose may be determined through clinical trials. In some embodiments, the compositions of this disclosure are formulated for intravenous administration.

[0080] Without further detailed description, it is believed that those skilled in the art can make full use of this disclosure based on the above description. Therefore, specific embodiments should be construed as merely illustrative and not in any way limiting the remainder of this disclosure, which is disclosed on pages 13 / 20 of CN 120826232 A.

[0081] Examples

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

[0083] Example 1: Constant natural killer T (iNKT) cell therapy in subjects with moderate acute respiratory distress syndrome secondary to viral infection

[0084] The following examples demonstrate that iNKT cell therapy as described herein effectively improves survival; reduces inflammatory response; 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 venous-venous extracorporeal membrane oxygenation (VV ECMO). In addition, iNKT cell therapy offers at least the following benefits: (i) exhibiting a favorable safety profile (e.g., no neurotoxicity or ≥ grade 3 TRAE observed); (ii) exhibiting transient persistence in the periphery, consistent with in vivo data describing the rapid translocation of iNKT cells from blood to tissues; (iii) opportunities for repeated administration (although alloantibodies were detected after iNKT cell administration and correlated with 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).

[0085] Invariant natural killer T (iNKT) cells, as major modulators of the immune response, make them an ideal immunotherapy.This disclosure relates to the use of ex vivo expanded allogeneic iNKT cells to treat viral diseases of the lungs for the treatment of a broad spectrum of diseases, including viral diseases of the lungs.

[0086] Without being bound by any particular theory, iNKT cells exert their antiviral effects through at least the following mechanisms: (i) direct viral killing: in a TCR-dependent manner by recognizing CD1d ligands in diseased tissues and activating them via a constant TCR; (ii) recruiting host immunity by recruiting host T cells and NK cells: in a TCR-independent manner by activating NK cells by recognizing stress signals via NK receptors (e.g., NKG2D, DNAM1); (iii) regulating and / or disrupting myeloid suppressor cells and inflammatory monocytes that protect the airway epithelium; (iv) recruiting and activating NK and T cells through cytokine secretion; (v) reversing T cell exhaustion, such as restoring the cytotoxic capacity, activation, and generation of partially exhausted T cells; (vi) inducing the maturation of immature dendritic cells; (vii) inhibiting pro-inflammatory 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 constant natural killer T (iNKT) cells in participants with moderate to severe acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 or influenza, whether intubated or at high risk of intubation, as determined using the Berlin definition. Part 1 employs a standard 3+3 dose escalation design for iNKT cells. All participants received a single infusion of iNKT cells. Participants also received other treatments and supportive care at the investigator's discretion. Once the maximum tolerated dose of iNKT cells was cleared in Part 1, an open expansion cohort was established. All participants were divided into three cohorts and received single infusions of different doses of iNKT cells: Cohort 1 received 100 × 10^6 iNKT cells; Cohort 2 received 300 × 10^6 iNKT cells; and Cohort 3 received 1000 × 10^6 iNKT cells.

[0088] Male and female subjects (aged > 18 years) with evidence of SARS-CoV-2 infection and diagnosed with 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 who had dose-limiting toxicities (DLT).

[0090] Secondary endpoints included: (i) time to extubation (maximum 30 days); (ii) mean daily sequential organ failure assessment score; (iii) change in C-reactive protein relative to baseline; (iv) decrease in quantitative viral load from upper and lower respiratory tract samples; (v) time from administration to viral clearance (maximum up to day 30); and (vi) number of participants who experienced viral reactivation and fungal infection.

[0091] A summary of the participant demographics by dose level cohort is shown in Table 1. The date of COVID-19 diagnosis for patients is shown in Figure 2 in the context of the SARS-CoV-2 strain epidemic in the United States. 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] Adverse reactions were assessed after iNKT cell therapy, 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 ≥ Grade 3 AE 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 ≥ Grade 3 TRAE 0 1 (25.0) 0 1 (5.0) Any TRAE leading to discontinuation of medication 0 0 0 0 Any TRAE leading to dose interruption 0 0 0 0 Any TRAE leading to death 0 0 0 0

[0098] Overall, iNKT cell therapy was well tolerated. Most of the observed adverse events (AEs) were consistent with a potential diagnosis of severe Covid-19 / ARDS requiring invasive mechanical ventilation (IMV). No dose-limiting toxicities were reported. Treatment-related 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 using Kaplan-Mayer survival curves, which showed a 70% survival rate in the study at 30 days post-treatment compared to a 10% survival rate in the control group at 30 days post-IMV initiation (Figure 3).

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

[0101] Table 3. Cohort-level incidence of concomitant infections. Specification 15 / 20 pages 19 CN 120826232 A

[0102]

[0103]

[0104] Cohort 3 (highest dose level) showed a reduced incidence of concomitant infections. Compared with the incidence in cohorts 1 and 2, an approximately 50% reduction in the overall reported incidence of 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% compared with the combined number of cohorts 1+2 (15% incidence compared to 71%). In contrast, the reported incidence of ventilator-associated pneumonia (VAP) in COVID ARDS ranges from 25% to 84%.

[0105] Cytokine release syndrome (CRS) may be a characteristic of acute respiratory distress syndrome (ARDS) secondary to viral infections (e.g., influenza or SARS-CoV-2). Therefore, key CRS markers, 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 (pre-infusion, single time point), an early post-infusion window (from 2 hours post-infusion on day 1 to day 7 post-infusion; D1–7) (corresponding to iNKT cell persistence measured in the periphery), and a late post-infusion window (days 10–28; D10–28). Data on ferritin, CRP, and D-dimer were collected on the day of treatment (D1, a single time point), in the early post-infusion window (days 2–8; D2–8), and in the late post-infusion window (days 8–28; D8–28). Dashed lines represent the upper limit of normal levels for the corresponding biomarkers in healthy individuals. Among the cytokines tested, IL-1α / β was undetectable or within the normal range (Figs. 4A–4B); and IL-6, ferritin, CRP, and D-dimer were elevated before infusion and did not increase after infusion (Figs. 4C–4F). The results indicate that iNKT cell therapy does not induce CRS.

[0106] Patients with severe ARDS can undergo venous-venous extracorporeal membrane oxygenation (VV ECMO), which may improve survival in these patients. However, mortality rates for ARDS patients undergoing VV ECMO remain high. In this study, patients undergoing VV ECMO treated with iNKT cells showed a 75% 90-day survival rate compared to control cases (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 tubing to an artificial lung in the machine, which increases oxygen and removes 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, as seen in mesenchymal stem cell therapy in ARDS patients undergoing ECMO, was observed.

[0107] After infusion of iNKT cells, the persistence of iNKT cells in the blood was assessed. The iNKT cells infused into the patient's PBMC were quantified by digital PCR based on donor-specific genetic markers. Each line represents data from one patient. Infused iNKT cells were detected up to day 6 post-infusion, with potentially lower levels of persistence at the highest dose levels persisting for a longer period, up to the last day of sampling on day 28 (Figures 6A to 6C). Peak levels of infused iNKT cells are shown in red. 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 prolonged detection of infused iNKT cells after infusion in ECMO patients was consistent with the absence of filter clogging in ECMO patients receiving iNKT cell therapy. The kinetics of iNKT cell tissue distribution in a mouse xenograft model showed rapid translocation of iNKT cells to tissues following intravenous injection (Fig. 6D). The observed persistence of iNKT cells after brief infusion in the patient's bloodstream was consistent with the kinetics of iNKT cell distribution from blood to tissue in vivo.

[0108] An increased anti-inflammatory response was observed following iNKT cell therapy infusion (Figs. 7A to 7R). Cytokines of particular interest are those known to play a key role in the pathogenesis of COVID-19, as shown in Figs. 7A, 7C, 7D, 7G, 7H, 7I, 7J, 7L, 7O, and 7P. (Data for IL-1 and IL-6 are shown in Figs. 4A to 4C). Among 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 pro-inflammatory 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 administration are indicated by an asterisk (*p<0.05; **p<0.01; data by one-way ANOVA analysis). Dashed lines represent the upper limit of normal levels for the corresponding biomarkers in healthy individuals.

[0109] Furthermore, chest X-rays showed improved lung function in patients within 24 hours after iNKT cell infusion compared to pre-infusion chest X-rays (Figures 10A-10B).Following iNKT cell infusion, patients experienced improved lung volume and parenchymal stability.

[0110] The presence of donor-specific alloantibodies (DSA) was determined on the day of administration and day 14. Figures 8A-8B indicate the induction of DSA following iNKT cell infusion. Patients were scored on day 14 post-infusion to determine whether DSA was induced. The incidence of DSA development decreased with increasing HLA class I matching but appeared to be independent of the degree of HLA class II matching. Serum DSA levels decreased post-administration with increasing HLA matching. DSA levels with concomitant MFI < 1,000 were considered negative and were not reported (Figure 8C). DSA levels were measured at discharge for 4 patients (patient discharge day, from left to right: 60, 28, 21, 28). DSA levels following 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 showed a 70% survival rate in the study, compared to 10% in the control group and 39% at CDC discharge.

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

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

[0114] The following examples demonstrate that iNKT cell therapy as described herein effectively improves survival; reduces pneumonia; and / or promotes anti-inflammatory responses in ARDS patients undergoing venous-venous extracorporeal membrane oxygenation (VV-ECMO).

[0115] Although mortality rates for severe COVID-19 respiratory failure have been trending toward improvement, primarily due to early corticosteroids and antiviral therapy, mortality remains high (47.9% to 84.4%). Increased venous-venous extracorporeal membrane oxygenation (VV-ECMO) support improved survival in a selected cohort of COVID-19 patients, but the overall 90-day mortality rate remained at 47%. VV-ECMO therapy has numerous complications, including bleeding, oxygenator failure, and hospital-acquired infections.For patients with severe COVID-19 respiratory failure, multimodal therapy is needed to enhance current interventions. Ideally, such therapy will modulate the initial excessive inflammatory response, but may also enhance the innate immune system against other hospital-acquired threats. Constant natural killer T (iNKT) cells comprise about 0.8% of white blood cells in healthy human hosts and are the natural home of damaged organs, including the lungs, where they suppress pro-inflammatory cytokines and protect epithelial tissues. Low circulating levels of iNKT cells may be a marker of mortality in COVID-19 ARDS (Kreutmair et al., Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia, Immunity. 2021 July 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)). IL-18 is a cytokine associated with the activation of unconventional T cells 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). Cells by MR1. Nature 422(6928):164–9. 10.1038 / nature01433). In addition, reduced IFNγ production was observed in circulating iNKT cells and iNKT cells.Among the remaining iNKTs in circulation, those expressing PD-1 and CD69 showed increased levels, while on day 15, high PD-1 expression persisted on iNKT cells from patients in the intensive care unit (Jouan et al., (2020) Phenotypical and functional alteration of unconventional T cells in severe 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 severe COVID-19 pneumonia and a mean age of 50 years met the established criteria for VV-ECMO cannulation. All four patients received dexamethasone and remdesivir, along with a single infusion of 1000 x 10⁶ iNKT cells. The primary endpoint was safety and tolerability, and several secondary and exploratory endpoints were identified, including all-cause mortality at 30 days.

[0117] No significant infusion-related events, including oxygenator failure, 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 the study drug. The persistence of circulating iNKT cells in the VV-ECMO cohort was comparable to that in study patients who did not undergo ECMO (Fig. 9B), and a reduced incidence of pneumonia was observed compared to the single-center VV-ECMO 2021–2022 cohort (n = 38). Notably, significantly increased levels of the anti-inflammatory cytokine IL-1-RA (Fig. 9C) and the growth factor VEGF-D were also observed.

[0118] iNKT cell therapy was well-tolerated in patients with severe COVID-19 ARDS who received VV-ECMO. The 30-day overall survival rate was 75%. Exploratory data, such as the rate of hospital-acquired infections and protective cytokine levels in treated patients, showed statistically significant correlations and potentially important trends.These early observations support the efficacy of iNKT cell-based immunotherapy in treating ARDS (e.g., COVID-19-related ARDS).

[0119] Other Embodiments

[0120] All features disclosed in this specification can be combined in any combination. Each feature of the disclosure in this specification (pages 18 / 20, 22 CN 120826232 A) can be replaced by an alternative feature serving the same, equivalent, or similar purpose. Therefore, unless explicitly stated otherwise, each disclosed feature 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 readily identify the essential features of this disclosure, and various changes and modifications can be made to adapt this disclosure to various uses and conditions without departing from the spirit and scope of this disclosure. Therefore, other embodiments are also within the scope of the claims.

[0122] Equivalents

[0123] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing functions and / or obtaining 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 invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications using the teachings of this invention. Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that embodiments of the invention may be practiced in ways different from those specifically described and claimed within the scope of the appended claims and their equivalents. The embodiments of the invention disclosed herein relate to each individual feature, system, article of manufacture, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of this disclosure if such features, systems, articles, materials, kits, and / or methods do not contradict each other.

[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 the ordinary meaning of defined terms. Definitions of terms are disclosed throughout the specification, including but not limited to the "Definitions" section.

[0125] Section headings are not intended to be construed as limiting the scope of this disclosure.

[0126] Unless clearly stated 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 “any one or both” of the elements so combined, i.e., elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements so combined. Other elements may optionally be present, whether related to or unrelated to those specifically identified by the “and / or” clause. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open-ended language such as “comprising,” a reference to “A and / or B” may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, 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 items are listed separately in a list, “or” or “and / or” should be interpreted as inclusive, that is, including several elements or at least one of the elements in the list, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate otherwise, such as “only one of…” or “exact one of…” or “consisting of…” when used in a claim, will refer to the inclusion of multiple elements or exactly one of the elements in the list. Generally, when preceded by an exclusive term (such as “any,” “one of,” “only one of,” or “exact one of”), the term “or” as used herein should be interpreted only as indicating an exclusive alternative (i.e., “one or the other, but not both”). When used in a claim, “consisting substantially of…” should have its ordinary meaning as used in the field of patent law.

[0129] As used herein in the specification and claims, the phrase “at least one” relating to a list having one or more elements should be understood to mean at least one element selected from any one or more elements in the element list, but not necessarily including at least one of every element specifically listed in the element list, and does not exclude any combination of elements in the element list. This definition also allows for the optional presence of elements other than those specifically identified in the element list referred to by the phrase “at least one”, whether related to or not related to those specifically identified elements.Therefore, 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, it refers to at least one, optionally including more than one B and no A (and optionally including elements other than A); in yet another embodiment, it refers to at least one, optionally including more than one A, and refers to at least one, optionally including more than one B (and optionally including other elements); and so on.

[0130] It should also be understood that, unless explicitly stated otherwise, 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 steps or order of the steps of the method described herein.

[0131] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “accommodating,” “constituting,” “made of,” etc., should be understood as open-ended, meaning including but not limited to. As set forth in Section 2111.03 of the Patent Examination Procedure Manual of the U.S. Patent Office, only the transitional phrases “composed of” and “substantially composed of” are closed or semi-closed transitional phrases, respectively. It should be understood that, in alternative embodiments, embodiments described in this document using open-ended transitional phrases (e.g., “comprising”) are also contemplated as “composed of the features described by the open-ended transitional phrase” and “substantially composed of.” For example, if this disclosure describes “a composition comprising A and B,” then this disclosure also contemplates alternative embodiments such as “a composition composed of A and B” and “a composition composed substantially of A and B.”Instruction Manual 20 / 20 Page 24 CN 120826232 A Figure 1 Figure 2 Instruction Manual Figure 1 / 21 Page 25 CN 120826232 A Figure 3 Figure 4A Instruction Manual Figure 2 / 21 Page 26 CN 120826232 A Figure 4B Figure 4C Instruction Manual Figure 3 / 21 Page 27 CN 120826232 A Figure 4D Figure 4E Instruction Manual Figure 4 / 21 Page 28 CN 120826232 A Figure 4F Figure 5 Instruction Manual Figure 5 / 21 Page 29 CN 120826232 A Figure 6A Figure 6B Instruction Manual Figure 6 / 21 Page 30 CN 120826232 A Figure 6C Figure 6D Instruction Manual Figure 7 / 21 Page 31 CN 120826232 A Figure 7A Figure 7B Instruction Manual Figure 8 / 21 Page 32 CN 120826232 A Figure 7C Figure 7D (Instruction Manual Appendix 9 / 21, Page 33, CN 120826232 A) Figure 7E Figure 7F (Instruction Manual Appendix 10 / 21, Page 34, CN 120826232 A) Figure 7G Figure 7H (Instruction Manual Appendix 11 / 21, Page 35, CN 120826232 A) Figure 7I Figure 7J (Instruction Manual Appendix 12 / 21, Page 36, CN 120826232 A) Figure 7K Figure 7L (Instruction Manual Appendix 13 / 21, Page 37, CN 120826232 A) Figure 7M Figure 7N (Instruction Manual Appendix 14 / 21, Page 38, CN 120826232 A) Figure 7O Figure 7P (Instruction Manual Appendix 15 / 21, Page 39, CN 120826232 A) Figure 7Q Figure 7R (Instruction Manual Appendix 16 / 21, Page 40, CN 120826232 A) Figure 8A Figure 8B (Instruction Manual Appendix 17 / 21, Page 41, CN) 120826232 A Figure 8C Figure 8D Instruction Manual Drawings 18 / 21 Page 42 CN 120826232 A Figure 9A Figure 9B Instruction Manual Drawings 19 / 21 Page 43 CN 120826232 A Figure 9C Figure 10A Instruction Manual Drawings 20 / 21 Page 44 CN 120826232 A Figure 10B Instruction Manual Drawings 21 / 21 Page 45 CN 120826232 A.

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.

Images