Diagnosis and treatment of cytokine release syndrome

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of cytokine release syndrome (CRS). The present invention also relates to methods for diagnosing patients suffering from CRS. The inventors investigate the role of iron homeostasis and CD44-mediated iron endocytosis in severe inflammatory responses and cytokine release syndrome, particularly in SARS-CoV-2 patients. The inventors demonstrate that iron endocytosis is upregulated in a CD44-dependent manner and CD44 protein levels increase during M1 macrophage activation. This effect is specific to M1 macrophages, while the levels of the canonical iron endocytic protein TfR1 / CD71 remain unchanged. Here, the inventors provide in vitro evidence for a direct role of CD44-mediated iron endocytosis in severe inflammatory responses, such as the cytokine release syndrome observed in SARS-CoV-2 patients. Thus, the present invention relates to antagonists of the CD44 / hyaluronic acid pathway for use in the treatment of cytokine release syndrome, particularly severe COVID-19 associated cytokine release syndrome, in a subject in need thereof.

Description

Technical Field

[0001] Field of the Invention The present invention relates to methods and pharmaceutical compositions for the treatment of cytokine release syndrome (CRS). The present invention also relates to methods of diagnosing patients suffering from cytokine release syndrome.

Background Art

[0002] Background of the Invention Recent studies have shown that cytokine release syndrome (CRS) is a major cause of morbidity and mortality in SARS-CoV-2 patients, and that elevated interleukin-6 (IL-6) levels are associated with respiratory failure (Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science. May 1, 2020; 368(6490): pp. 473-474. doi: 10.1126 / science.abb8925). Furthermore, it has been shown that CD44 / hyaluronate (HA) regulates chemokine gene expression in alveolar macrophages (McKee et al., Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J Clin Invest. November 15, 1996; 98(10):2403-13. DOI: 10.1172 / JCI1 19054). Furthermore, ferritin levels have been shown to be highly upregulated in severely ill SARS-CoV-2 patients (Zhou et al., Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China, a retrospective cohort study. The Lancet (2020) DOI: https: / / doi.org / 10.1016 / S0140-6736(20)30566-3), which suggests a causal role of iron in the physiological and pathological aspects of Covid-19.

[0003] Perricone et al. (Authorea, Inc, DOI: 10.22541 / au.158880283.3460 4328) discuss the role of iron removal therapy in the context of COVID-19 as part of hyperferritinemia syndrome. Dalamaga et al. (2020, Metabolism, Clinical and experimental, 108, 154260) discuss the possible usefulness of iron chelators as adjuncts to treatment regimens for COVID-19.

[0004] The inventors have recently elucidated the functional role of CD44, a plasma membrane receptor for Hyal, in regulating epithelial-mesenchymal transition, cancer progression, immune responses, and inflammation. In particular, the inventors discovered that CD44 mediates the endocytosis of iron-bound Hyal in cancer cells and immune T cells. In connection with this, the inventors have shown that iron plays a central role in regulating epigenetic plasticity and cellular identity, and as a result acts as a metal catalyst to promote the demethylation of inhibitory histone marks H3K9me2 and H3K27me3. In the course of this research, the inventors found that the gene encoding the IL6 receptor (IL6R / CD126) is one of the top iron-regulating genes, and in this case, iron—and therefore CD44 / Hyal—mediates the depletion of H3K9me2, leading to the upregulation of IL6R. Importantly, the inventors also demonstrated that an anti-CD44 antibody can block iron endocytosis and consequently counteract this effect. This study has generated considerable international interest in a wide range of scientific communities, including basic researchers and clinicians (Muller et al., CD44 regulates epigenetic plasticity by mediating iron endocytosis, BioRxiv, DOI: https: / / doi.org / 10.1101 / 693424, accepted by Nature Chemistry).

[0005] In this disclosure, the inventors demonstrate that iron endocytosis is upregulated in a CD44-dependent manner during the activation process of M1 macrophages, and that CD44 protein levels are elevated. This effect is specific to M1 macrophages, while the levels of the canonical iron endocytosis protein TfR1 / CD71 remain unchanged. Taken together, these data suggest a direct role of CD44-mediated iron endocytosis in the severe inflammatory response observed in SARS-CoV-2 patients.

[0006] Furthermore, there is no disclosure in the art regarding the use of CD44 / hyaluronic acid (HA) pathway antagonists in the treatment of cytokine release syndrome (CRS), particularly in SARS-CoV-2 patients. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2008 / 144890 [Patent Document 2] International Publication No. 0139815 [Patent Document 3] International Publication No. 2004 / 018000 [Patent Document 4] International Publication No. 2005 / 049082 [Patent Document 5] International Publication No. 2008 / 079246 [Patent Document 6] International Publication No. 2014 / 023869 [Patent Document 7] International Publication No. 2005 / 087264 [Patent Document 8] International Publication No. 2014 / 198843 [Patent Document 9] International Publication No. 2011 / 095498 [Patent Document 10] International Publication No. 2013 / 063498 [Patent Document 11] International Publication No. 2007 / 039761 [Patent Document 12] International Publication No. 2015 / 097170 [Patent Document 13] U.S. Patent No. 4,816,567 [Patent Document 14] U.S. Patent No. 5,225,539 [Patent Document 15] U.S. Patent No. 5,585,089 [Patent Document 16] U.S. Patent No. 5,693,761 [Patent Document 17] U.S. Patent No. 5,693,762 [Patent Document 18] U.S. Patent No. 5,859,205 [Patent Document 19] International Publication No. 90 / 07861 [Patent Document 20] U.S. Patent No. 5,591,669 [Patent Document 21] U.S. Patent No. 5,598,369 [Patent Document 22] U.S. Patent No. 5,545,806 [Patent Document 23] U.S. Patent No. 5,545,807 [Patent Document 24] U.S. Patent No. 6,150,584 [Patent Document 25] U.S. Patent No. 5,565,332 [Patent Document 26] U.S. Patent No. 5,573,905 [Patent Document 27] U.S. Patent No. 5,229,275 [Patent Document 28] U.S. Patent No. 5,567,610 [Patent Document 29] U.S. Patent No. 5,800,988 [Patent Document 30] U.S. Patent No. 5,874,541 [Patent Document 31] U.S. Patent No. 6,015,695 [Patent Document 32] U.S. Patent No. 6,765,087 [Patent Document 33] U.S. Patent No. 6,838,254 [Patent Document 34] U.S. Patent No. 6,566,135 [Patent Document 35] U.S. Patent No. 6,566,131 [Patent Document 36] U.S. Patent No. 6,365,354 [Patent Document 37] U.S. Patent No. 6,410,323 [Patent Document 38] U.S. Patent No. 6,107,091 [Patent Document 39] U.S. Patent No. 6,046,321 [Patent Document 40] U.S. Patent No. 5,981,732 [Patent Document 41] U.S. Patent No. 6,573,099 [Patent Document 42] U.S. Patent No. 6,506,559 [Patent Document 43] International Publication No. 01 / 36646 [Patent Document 44] International Publication No. 99 / 32619 [Patent Document 45] International Publication No. 01 / 68836 [Patent Document 46] Chinese Patent No. 104561000 [Patent Document 47] International Publication No. 0149266 [Patent Document 48] International Publication No. 2009103727 [Patent Document 49] International Publication No. 2011109521 [Patent Document 50] International Publication No. 2014059417 [Patent Document 51] International Publication No. 2017153475

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Non-licensed literature

[0008] [Non-licensed document 1] Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science. May 1, 2020; 368(6490): 473-474. doi: 10.1126 / science.abb8925 [Non-licensed document 2] McKee, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J Clin Invest. 1996 Nov 15;98(10):2403~13 pp. DOI: 10.1172 / JCI1 19054 [Non-licensed document 3] Zhou ら, Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China, a retrospective cohort study. The Lancet (2020) DOI: https: / / doi.org / 10.1016 / S0140-6736(20)30566-3

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[0009] Summary of the present invention This invention relates to methods and pharmaceutical compositions for the treatment of cytokine release syndrome (CRS). The invention also relates to methods for diagnosing patients suffering from cytokine release syndrome (CRS). In particular, the invention is defined by the claims.

[0010] In one aspect of this disclosure, the present invention relates to a CD44 / hyaluronic acid pathway antagonist or a pharmaceutical composition comprising the same for use in the treatment of cytokine release syndrome in subjects requiring its use, wherein the antagonist or pharmaceutical composition comprising the CD44 / hyaluronic acid pathway antagonist is selected from the group consisting of CD44-targeting compounds, which is selected from the group consisting of CD44 antagonists and CD44 expression inhibitors. The present invention further relates to the use of CD44-targeting compounds, selected from the group consisting of CD44 antagonists and CD44 expression inhibitors, for the manufacture of a pharmaceutical for the treatment of cytokine release syndrome in subjects requiring its use. The present invention also relates to a method for the treatment of cytokine release syndrome in subjects requiring its use, which comprises the steps of administering a therapeutically effective amount of a CD44-targeting compound, selected from the group consisting of CD44 antagonists and CD44 expression inhibitors, thereby preventing or mitigating cytokine release syndrome.

[0011] In certain aspects, cytokine release syndrome is a severe form of COVID-19-related cytokine release syndrome.

[0012] If necessary, the CD44 antagonist is selected from the group consisting of small organic molecules, polypeptides, aptamers, or antibodies. For example, the antibody is selected from the group consisting of RG7356 and vivacuzumab.

[0013] If necessary, the CD44 expression inhibitor is selected from the group consisting of antisense oligonucleotides, shRNA, siRNA, RNAi, and ribozymes. [Modes for carrying out the invention]

[0014] Detailed description of the present invention The inventors investigate the roles of iron homeostasis and CD44-mediated iron endocytosis in severe inflammatory responses and cytokine release syndrome (CRS), particularly in SARS-CoV-2 patients. The inventors demonstrate that iron endocytosis is upregulated in a CD44-dependent manner and that CD44 protein levels increase during M1 macrophage activation. This effect is specific to M1 macrophages, while the levels of the canonical iron endocytosis protein TfR1 / CD71 remain unchanged. In this invention, the inventors provide in vitro evidence for the direct role of CD44-mediated iron endocytosis in severe inflammatory responses, such as cytokine release syndrome (CRS) observed in SARS-CoV-2 patients. In summary, the present invention highlights the role of CD44-mediated iron endocytosis in severe inflammatory responses and cytokine release syndrome (CRS), the potential use of CD44 / hyaluronic acid (HA) pathway antagonists, and the targeting of iron homeostasis in the treatment of cytokine release syndrome (CRS), particularly in SARS-CoV-2 patients. The present invention also identifies CD44 as a novel marker of predisposition to severe COVID-19.

[0015] Accordingly, this disclosure relates to targeting iron homeostasis in the treatment of cytokine release syndrome (CRS), particularly severe COVID-19-associated CRS. The invention also relates to targeting iron homeostasis in the treatment of adult respiratory distress syndrome (ARDS), particularly severe COVID-19-associated ARDS. The invention also relates to targeting iron homeostasis in the treatment of macrophage activation syndrome (MAS), particularly severe COVID-19-associated MAS. The invention also relates to targeting iron homeostasis in the treatment of iron-related inflammatory diseases and alveolar inflammatory responses, particularly in patients with COVID-19. The invention also relates to targeting iron homeostasis in the treatment of COVID-19, particularly severe COVID-19.

[0016] Treatment method In a first aspect, the present invention relates to a CD44 / hyaluronic acid (HA) pathway antagonist for use in the treatment of cytokine release syndrome (CRS) in subjects requiring it.

[0017] In some embodiments, the present invention relates to a CD44 / hyaluronic acid (HA) pathway antagonist for use in the treatment of severe COVID-19-associated CRS in subjects requiring it.

[0018] In a further embodiment, the present invention relates to a CD44 / hyaluronic acid (HA) pathway antagonist for use in the treatment of respiratory distress syndrome (ARDS) in subjects requiring it.

[0019] In a further embodiment, the present invention relates to a CD44 / hyaluronic acid (HA) pathway antagonist for use in the treatment of macrophage activation syndrome (MAS) in subjects requiring it.

[0020] In a further embodiment, the present invention relates to a CD44 / hyaluronic acid (HA) pathway antagonist for use in the treatment of iron-related inflammatory diseases and alveolar inflammatory reactions in subjects requiring such treatment.

[0021] In a further aspect, the present invention relates to an antagonist of the CD44 / hyaluronic acid (HA) pathway for use in the treatment of COVID-19, particularly severe COVID-19, in subjects requiring it.

[0022] As used herein, the terms “subject,” “individual,” or “patient” are interchangeable and refer to mammals. Typically, the subject according to the present invention refers to any subject, preferably a human. In certain embodiments, the term “subject” refers to a subject who has or is at risk of developing cytokine release syndrome (CRS), particularly COVID-19-related CRS. In certain embodiments, the term “subject” refers to a subject who has or is at risk of developing adult respiratory distress syndrome (ARDS), particularly severe COVID-19-related ARDS. In certain embodiments, the term “subject” refers to a subject who has or is at risk of developing macrophage activation syndrome (MAS), particularly severe COVID-19-related MAS. In certain embodiments, the term “subject” refers to a subject who has or is at risk of developing iron-related inflammatory disease and alveolar inflammatory response, particularly iron-related inflammatory disease and alveolar inflammatory response in COVID-19 patients. In certain embodiments, the term “subject” refers to a subject who has or is at risk of contracting COVID-19, particularly severe COVID-19.

[0023] As used herein, the terms “treatment” or “to treat” include both prophylactic or preventive treatments and curative or disease-modifying treatments, including treatments for subjects at risk of developing or suspected of having the disease, and subjects diagnosed with the disease or a diseased or medical condition, and the term includes the suppression of clinical relapses. Treatments may be administered to subjects with a medical disability or at risk of ultimately acquiring a disability for the following purposes: to prevent, cure, or delay the onset of one or more symptoms of the disability or recurrent disability, to reduce their severity, to induce remission, or to extend the survival of the subject beyond the survival expected in the absence of such treatment. “Treatment regimen” means a pattern of treatment for a disease, e.g., a pattern of medication used in the course of treatment. Treatment regimens may include induction regimens and maintenance regimens. The terms “induction regimen” or “induction period” mean a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general goal of an induction regimen is to provide the patient with a high level of medication during the initial phase of the treatment regimen. An induction regimen may (partially or entirely) be a “loading regimen,” which may include administering a higher dose of medication than the physician would expect to use in the maintenance regimen, administering the medication more frequently than the physician would expect to use in the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain a subject in remission during the treatment of a disease, for example, over a long period (several months or several years). A maintenance regimen may be a continuous therapy (e.g., administering medication at regular intervals, e.g., once a week, once a month, once a year, etc.) or an intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment at the time of relapse, or treatment when certain predetermined criteria [e.g., manifestation of the disease] are reached).

[0024] As used herein, the terms “cytokine release syndrome” or “CRS” have their general meaning in the art and refer to a disease caused by the massive and rapid release of cytokines from immune cells into the bloodstream. The terms “cytokine release syndrome” or “CRS” also refer to a systemic inflammatory response observed following antibody administration and adoptive T-cell therapy, or a cytokine response occurring in infectious diseases such as coronavirus disease 2019 (COVID-19), sepsis, Ebola, avian influenza, and non-infectious diseases such as graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), and systemic inflammatory response syndrome (SIRS) (Shimabukuro-Vornhagen et al., Cytokine release syndrome. J Immunother Cancer. June 15, 2018; 6(1):56. doi:10.1186 / s40425-018-0343-9). The term "cytokine release syndrome" or "CRS" refers to a condition that may occur after treatment with certain types of immunotherapy, such as antibodies or CAR-T cells, as described in the National Cancer Institute (NCI) Dictionary of Cancer Terms, and is caused by the massive and rapid release of cytokines into the bloodstream from immune cells affected by immunotherapy. CRS is the most common acute toxicity of CAR T cells, and CRS is associated with a progressive systemic inflammatory process and is characterized by high fever, hypotension, hypoxia, neurotoxicity, and / or multi-organ toxicity (Davila et al., Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. February 19, 2014;6(224):224ra25. doi: 10.1126 / scitranslmed).The severity of CRS ranges from mild symptoms such as fever, muscle pain, and fatigue to severe symptoms, including, but not limited to, acute respiratory distress syndrome (ARDS), hypotension, disseminated intravascular coagulation, renal and hepatotoxicity, organ dysfunction, cytokine profiles such as elevated IL-6, IL-2R, and IL-1 beta, and / or neurotoxicity. (Shalabi et al., Novel Designs of Early Phase Trials for Cancer Therapeutics. Chapter 12 - Cell-Based Therapies: A New Frontier of Personalized Medicine. Editors: Shivaani Kummar, Chris Takimoto. Academic Press, 2018, pp. 175-191, ISBN 9780128125120, doi.org / 10.1016 / B978-0-12-812512-0.00012-9; Maude et al., Managing cytokine release syndrome associated with novel T cell-engaging) Therapies. Cancer J. March-April 2014;20(2): pp. 119-122. doi: 10.1097 / PPO.0000000000000035). Cytokine release syndrome may also be associated with findings of macrophage activation syndrome (MAS) / hemophagocytic lymphohistiocytosis (HLH). The term "cytokine release syndrome" also refers to COVID-related CRS, particularly severe COVID-19-related CRS. Recent studies have shown that cytokine release syndrome (CRS) is a major cause of morbidity and mortality in SARS-CoV-2 patients, and that elevated IL-6 levels are associated with respiratory failure (Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science. May 1, 2020; 368(6490): pp. 473-474. doi: 10.1126 / science.abb8925).

[0025] As used herein, the terms “adult respiratory distress syndrome” or “ARDS” have their general meaning in the art and refer to an acute and severe inflammatory pneumonia process caused by pulmonary or systemic trauma, most commonly initiated by pneumonia, infection, sepsis, or trauma (Ranieri et al., ARDS Definition Task Force. Acute respiratory distress syndrome: the Berlin Definition. JAMA. June 20, 2012; 307(23): 2526-33. doi: 10.1001 / jama.2012.5669; Hernandez-Beeftink et al., Genomics and the Acute Respiratory Distress Syndrome: Current and Future Directions. Int J Mol Sci. August 16, 2019; 20(16). pii: E4004. doi: 10.3390 / ijms20164004). The term “adult respiratory distress syndrome” or “ARDS” also refers to life-threatening lung injury that results in fluid leakage into the lungs, making breathing difficult and preventing oxygen from being taken into the body, as described by The American Lung Association (lung.org / lung-health-diseases). The term “adult respiratory distress syndrome” or “ARDS” also refers to respiratory failure, which is characterized by: signs and symptoms, e.g., severe shortness of breath, forced and abnormal tachypnea, hypotension, confusion and extreme fatigue, and the presence of underlying heart or lung disease, as described by the Mayo Clinic College of Medicine and Science (mayoclinic.org / patient-care-and-health-information). The most common underlying causes of ARDS include infection, sepsis, inhalation of harmful substances, pancreatitis, trauma, pneumonia, massive blood transfusion and burns. The pathophysiology of ARDS is due to acute inflammation of the alveolar spaces and a decrease in normal gas exchange.Fibrin deposition in the alveolar space and lung parenchyma is consistently observed with ARDS and contributes to hyaline membrane formation and subsequent alveolar fibrosis. This promotes the onset and progression of respiratory dysfunction and right heart failure (Whyte CS, Morrow GB, Mitchell JL, Chowdary P, Mutch NJ. Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat COVID-19. J Thromb Haemost. April 23, 2020. doi: 10.1111 / jth.14872). The term “adult respiratory distress syndrome” or “ARDS” also refers to severe COVID-19-related ARDS.

[0026] As used herein, the terms “macrophage activation syndrome” or “MAS” have their general meaning in the art and refer to a disorder characterized by pancytopenia, hepatic failure, coagulation disorders, and neurological symptoms, which is caused by the activation and uncontrolled proliferation of T lymphocytes and well-differentiated macrophages, resulting in widespread hemophagocytosis and cytokine overproduction. The term "macrophage activation syndrome" or "MAS" also refers to a life-threatening complication of rheumatic disease that occurs more frequently in individuals with systemic juvenile idiopathic arthritis (SJIA) and, even more frequently, in individuals with adult-onset Still's disease (Crayne et al., The Immunology of Macrophage Activation Syndrome. Front Immunol. February 1, 2019; 10:119. doi:10.3389 / fimmu.2019.00119; Lerkvaleekul and Vilaiyuk. Macrophage activation syndrome: early diagnosis is key. Open Access Rheumatol. August 31, 2018; 10:117-128. doi: 10.2147 / OARRR.S151013). The term "macrophage activation syndrome" also refers to severe COVID-19-associated MAS (Merad and Martin. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat. Rev. Immunol. May 6, 2020. doi: 10.1038 / s41577-020-0331-4).

[0027] As used herein, the term “iron-related inflammatory disease” has its general meaning in the art and refers to inflammatory diseases associated with dysfunction of iron homeostasis. In some embodiments, the term “iron-related inflammatory disease” refers to inflammatory diseases associated with iron overload syndrome. The term “iron-related inflammatory disease” also refers to severe iron-related inflammatory responses, particularly severe iron-related inflammatory responses in COVID-19 patients.

[0028] As used herein, the term “alveolar inflammatory response” has a general meaning in the art and refers to inflammatory diseases associated with dysfunction of alveolar macrophages, for example, after chest trauma, ischemia / reperfusion, hemorrhagic shock, and burns. The term “alveolar inflammatory response” also refers to inflammatory diseases associated with increased chemokine gene expression in alveolar macrophages in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (Niesler et al., Role of alveolar macrophages in the inflammatory response after trauma. Shock. July 2014;42(l):3-10. doi: 10.1097 / SHK.0000000000000167). The term “alveolar inflammatory response” also refers to COVID-19-related alveolar inflammatory responses.

[0029] As used herein, the terms “COVID-19” or “coronavirus disease 2019” have their common meaning in the art and refer to the infectious coronavirus disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a coronavirus newly identified in Wuhan, China in December 2019. The term “COVID-19” also refers to 2019-nCoV acute respiratory disease. COVID-19 results in mild to moderate respiratory illness, but in some cases it can develop into severe COVID-19.

[0030] As used herein, the term “severe COVID-19” has its general meaning in the art and refers to the side effects of COVID-19 resulting in severe respiratory illness, pneumonia, viral sepsis, cytokine release syndrome (CRS), acute respiratory distress syndrome (ARDS), macrophage activation syndrome (MAS), and multi-organ failure syndrome caused by an increased inflammatory response, such as: renal and pulmonary failure, respiratory failure, arterial inflammation, myocarditis (also known as inflammatory cardiomyopathy), myocardial injury, thrombosis, venous thromboembolism, and cardiovascular disease. (Han Y, Zeng H, Jiang H, Yang Y, Yuan Z, Chen X, Jing Z, Liu B, Chen J, Nie S, Zhu J, Li F, Ma C. CSC Expert Consensus on Principles of Clinical Management of Patients with Severe Emergent Cardiovascular Diseases during the COVID-19 Epidemic. Circulation. March 27, 2020.) These are as described in doi: 10.1161 / CIRCULATIONAHA.120.047011, and include pulmonary embolism, neurotoxicity, Kawasaki disease (also known as mucocutaneous lymphadenopathy), and cutaneous manifestations of COVID-19, as described in (Sachdeva M, Gianotti R, Shah M, Lucia B, Tosi D, Veraldi S, Ziv M, Leshem E, Dodiuk-Gad RP. Cutaneous manifestations of COVID-19: Report of three cases and a review of literature. J Dermatol Sci. April 29, 2020. pii: S0923-1811(20)30149-3. doi: 10.1016 / j.jdermsci.2020.04.011).

[0031] The term "CD44" has a general meaning in the art and refers to the transmembrane glycoprotein CD44 molecule (Indian blood type) involved in cell-cell interactions, cell adhesion, and migration. As used herein, the term "CD44" is intended to refer to the transmembrane glycoprotein, hyaluronic acid (HA or Hyal) plasma membrane receptor, which is involved in a wide variety of physiological and pathological processes, including development, inflammation, immune responses, wound healing, regulation of epithelial-mesenchymal transition (EMT), and cancer progression (Ponta, H., Sherman, L. & Herrlich, PA CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 4, pp. 33-45, (2003); Zoller, M. CD44: can a cancer-initiating cell profit from an abundantly expressed molecule? Nat. Rev. Cancer 11, pp. 254-267, (2011)). The term "CD44" also refers to a cell surface glycoprotein, whose isomer has the GeneCards Identifier GC11P035139 and the UniProt Knowledgebase UniProtKB / Swiss-Prot database identifier P16070 (Stelzer et al., The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. June 20, 2016; 54:1.30.1~1.30.33. doi: 10.1002 / cpbi.5; UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. January 8, 2019; 47(D1):D506~D515. doi: 10.1093 / nar / gky1049).CD44 interacts with hyaluronate (Hyal), a class of glycosaminoglycan biomolecules, and mediates its endocytosis (Aruffo, A., Stamenkovic, I, Melnick, M., Underhill, CB & Seed, B. CD44 is the principal cell surface receptor for hyaluronate. Cell 61, pp. 1303-1313, (1990); Hua, Q., Knudson, CB & Knudson, W. Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J. Cell Sci. 106, pp. 365-375, (1993)).

[0032] The terms "hyaluronic acid," "HA," or "Hyal" have a common meaning in the art and refer to hyaluronic acid (HA), also known as hyaluronate (Hyal) and hyaluronan. As used herein, the terms “hyaluronic acid” or “HA” are intended to refer to a class of glycosaminoglycan biomolecules, which are involved in a wide variety of cellular functions, including cell proliferation, cell migration, epithelial-mesenchymal transition (EMT), and cancer progression (Aruffo, A., Stamenkovic, I, Melnick, M., Underhill, CB & Seed, B. CD44 is the principal cell surface receptor for hyaluronate. Cell 61, pp. 1303-1313, (1990); Hua, Q, Knudson, CB & Knudson, W. Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J. Cell Sci. 106, pp. 365-375, (1993)).

[0033] As used herein, the term “CD44 / hyaluronic acid (HA) pathway antagonist” refers to any compound selected from the group consisting of, but not limited to, compounds targeting iron homeostasis, CD44-targeting compounds selected from the group consisting of CD44 antagonists or CD44 expression inhibitors, compounds targeting hyaluronic acid (HA), inhibitors of hyaluronic acid (HA) / iron internalization, inhibitors of iron endocytosis, iron chelators, e.g., deferoxamine (DFO); deferoxamine mesylate; deferoxamine hydrochloride; deferasirox (DFX); deferipron (DFP); deferritrin; deferipon; metformin; metformin; deferritazole; hepcidine fragment; and siderophores. CD44 / hyaluronic acid (HA) pathway antagonists exert their therapeutic effects by antagonizing IL6 signaling.

[0034] The term "CD44 antagonist" has its general meaning in the art and refers to a compound that selectively inactivates CD44. The term "CD44 antagonist" refers to any compound that can directly or indirectly inhibit the signaling cascade associated with CD44, inhibit CD44 ligand binding (hyaluronic acid (HA)), inhibit iron binding to the CD44 / HA complex, inhibit endocytosis of the iron-bound CD44 / HA complex, inhibit internal translocation of iron-bound hyaluronic acid, subsequently inhibit demethylation of the inhibitory histone marks H3K9me2 and H3K27me3, inhibit H3K9me2 depletion, and inhibit the upregulation of iron-regulating genes (e.g., IL6 receptor (IL6R / CD126)). As used herein, the term “selectively inactivates” refers to a compound that preferentially inactivates CD44 with greater affinity and potency, respectively, than its interactions with other subtypes or isoforms of the CD44 signaling family of receptors and cell surface glycoproteins. A CD44 antagonist also refers to a compound that reduces CD44 activity levels. Compounds that preferentially inactivate CD44 but can also inactivate other CD44 signaling family members of receptor subtypes and cell surface glycoproteins are intended as partial or complete antagonists. Typically, CD44 antagonists are small organic molecules, polypeptides, aptamers, or antibodies.

[0035] The CD44 antagonist is well known in the art, as described in International Publication Nos. 2008 / 144890; International Publication No. 0139815; International Publication Nos. 2004 / 018000; International Publication Nos. 2005 / 049082; International Publication Nos. 2008 / 079246; International Publication Nos. 2014 / 023869; and International Publication Nos. 2005 / 087264.

[0036] Tests and assays for determining whether a compound is a CD44 antagonist are well known in the art, as described in International Publication No. 2014 / 198843; International Publication No. 2008 / 144890; International Publication No. 2011 / 095498; International Publication No. 2013 / 063498; International Publication No. 2004 / 018000; International Publication No. 2005 / 049082; International Publication No. 2008 / 079246; International Publication No. 2005 / 087264; International Publication No. 2014 / 023869; International Publication No. 2007 / 039761 "CRYSTAL STRUCTURE OF CD44 AND ITS USE"; Weigand et al., 2012; D'Arena et al., 2014.

[0037] In some embodiments, the CD44 antagonist is an anti-CD44 antibody selected from the group consisting of RG7356 anti-CD44 antibodies, but is not limited to: vivacuzumab; the antibodies described in International Publication No. 2008 / 144890; International Publication No. 2004 / 018000; International Publication No. 2005 / 049082; International Publication No. 2008 / 079246; and International Publication No. 2005 / 087264.

[0038] In some embodiments, the CD44 antagonist is a peptide, or a CD44 ligand selected from the ligands described in International Publication No. 2015 / 097170, but is not limited to a peptide.

[0039] In some embodiments, the antibodies of the present invention compete for binding to the above-mentioned CD44 (and hyaluronic acid (HA)) antibodies.

[0040] In another embodiment, the compounds of the present invention are aptamers. Aptamers are a class of molecules that serve as substitutes for antibodies in terms of molecular recognition. Aptamers are oligonucleotide sequences that possess the ability to recognize virtually any class of target molecules with high affinity and specificity. Such ligands can be isolated through in vitro evolution of random sequence libraries (SELEX), as described in Turek C and Gold L, 1990. Random sequence libraries are available by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of a unique sequence that is ultimately chemically modified. Possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena SD, (1999). Peptide aptamers consist of a conformationally constrained antibody variable region presented by a platform protein, for example, by Escherichia coli (E. coli) thioredoxin A selected from a combinatorial library by a two-hybrid method (Colas et al., 1996). Subsequently, after the aptamers directed toward the target of the present invention are produced as described above, those skilled in the art can easily select those that block or inactivate the target.

[0041] In another embodiment, the compound of the present invention is an antibody directed against a target (a term including the “antibody portion”).

[0042] In one embodiment of the antibody or a portion thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the light chain of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the heavy chain of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the Fab portion of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the F(ab')2 portion of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the Fc portion of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the Fv portion of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes the variable domain of the antibody. In one embodiment of the antibody or a portion thereof described herein, the antibody portion includes one or more CDR domains of the antibody.

[0043] As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. In particular, "antibody" includes polyclonal antibodies and monoclonal antibodies, as well as their monovalent and bivalent fragments. Furthermore, "antibody" includes chimeric antibodies, fully synthetic antibodies, single-chain antibodies, and their fragments. Antibodies may be human antibodies or non-human antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0044] Antibodies are prepared according to conventional methodologies. Monoclonal antibodies can be produced using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in this invention, mice or other suitable host animals are immunized with the target antigen type at appropriate intervals (e.g., twice a week, once a week, twice a month, or once a month). The final "booster immunization" antigen can be administered to the animals within one week of sacrifice. It is often desirable to use immunological adjuvants in the immunization process. Suitable immunological adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or QuilA, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the art. Animals can be immunized subcutaneously, intraperitoneally, intramuscularly, intravenously, intranasally, or through other routes. Multiple routes allow for the immunization of a given animal with multiple forms of antigens.

[0045] In short, the antigen can be provided as a synthetic peptide corresponding to the desired antigenic region in the target. Following the immunotherapy regimen, lymphocytes are isolated from the spleen, lymph nodes, or other organs of an animal and fused with a suitable myeloma cell line using an agonist, such as polyethylene glycol, to form hybridomas. Following fusion, the cells are placed in a culture medium in which the hybridomas, rather than the fusion partners, can grow, using a standard method such as that described in (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following the culture of the hybridomas, the cell supernatant is analyzed for the desired specificity, i.e., the presence of antibodies that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and Western blotting. Other screening techniques are well known in the art. Preferred techniques include those that confirm the binding of antibodies to conformationally intact and naturally folded antigens, such as undenatured ELISA, flow cytometry, and immunoprecipitation.

[0046] Importantly, as is well known in this field, only a small fraction of antibody molecules have paratopes involved in antibody binding to their epitopes (for the whole, see Clark, WR (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th edition, Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors in the complement cascade but are not involved in antigen binding. Antibodies in which the pFc' region is enzymatically cleaved, or antibodies produced without the pFc' region, are called (F(ab')2 fragments) and retain both antigen-binding sites of the intact antibody. Similarly, antibodies in which the Fc region is enzymatically cleaved, or antibodies produced without the Fc region, are called Fab fragments and retain one of the antigen-binding sites of the intact antibody molecule. Further, a Fab fragment consists of a covalently bound antibody light chain and a portion of the antibody heavy chain called Fd. The Fd fragment is the primary determinant of antibody specificity (a single Fd fragment can associate with up to 10 different light chains without altering antibody specificity), and the Fd fragment alone retains epitope-binding ability.

[0047] Within the antigen-binding region of an antibody, as is well known in this art, there is a complementation-determining region (CDR) that directly interacts with the antigen's epitope and a framework region (FR) that maintains the paratope's tertiary structure (see Clark, 1986; Roitt, 1991 for the whole picture). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulin, there are four framework regions (FR1 to FR4) separated by three complementation-determining regions (CDR1 to CDRS). The CDR, and especially the CDRS region, particularly the heavy chain CDRS, plays a major role in antibody specificity.

[0048] It is now well established in the art that the non-CDR region of a mammalian antibody can be replaced with a similar region of an allospecific or heterospecific antibody while preserving the epitope specificity of the original antibody. This is most clearly demonstrated in the development and use of "humanized" antibodies, in which case the non-human CDR is covalently bound to the human FR region and / or Fc / pFc' region to produce a functional antibody.

[0049] The present invention provides compositions and methods comprising humanized antibodies in certain embodiments. As used herein, “humanized” means an antibody in which some, most, or all of the amino acids outside the CDR region are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Patents 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are incorporated herein by reference. Furthermore, U.S. Patents 5,585,089 and 5,693,761, as well as International Publication No. 90 / 07861, propose four possible criteria that can be used in the design of humanized antibodies. The first proposal was to use, in the case of acceptors, a framework derived from a specific human immunoglobulin that is normally homologous to the donor immunoglobulin being humanized, or a consensus framework derived from multiple human antibodies. The second proposal was that if a certain amino acid in the human immunoglobulin framework is not ordinary, but the donor amino acid at that position is typical in the human sequence, then the donor amino acid can be selected instead of the acceptor. The third proposal was that the donor amino acid can be selected instead of the acceptor amino acid at positions directly adjacent to three CDRs in the humanized immunoglobulin chain. The fourth proposal was to use the donor amino acid residue at a framework position in the three-dimensional model of the antibody where the amino acid is predicted to have a side chain atom within 3A of the CDR and is predicted to be able to interact with the CDR. The above methods are merely examples of some methods that can be used by those skilled in the art to produce humanized antibodies. Those skilled in the art will be familiar with other antibody humanization methods.

[0050] In one embodiment of the humanized form of an antibody, some, most, or all of the amino acids outside the CDR region are replaced with amino acids derived from human immunoglobulin molecules, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not impair the antibody's ability to bind to a given antigen. Suitable human immunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. The “humanized” antibody retains similar antigen specificity to the original antibody. However, using certain methods relating to humanization, the affinity and / or specificity of antibody binding can be improved using “directed evolution” methods, as described by Wu et al., / . Mol. Biol. 294:p. 151, 1999, the details of which are incorporated herein by reference.

[0051] Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice with respect to most of the human immunoglobulin heavy and light chain loci. See, for example, U.S. Patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584 and the references cited herein, the contents of which are incorporated herein by reference. These animals are genetically modified and therefore have functional deletions in the production of endogenous (e.g., mouse) antibodies. These animals can be further modified to contain all or part of the human germline immunoglobulin loci, and as a result, immunization of these animals will result in the production of fully human antibodies against the target antigen. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma techniques. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not induce a human anti-mouse antibody (KAMA) response when administered to humans.

[0052] In vitro methods for producing human antibodies also exist. These include phage display technology (U.S. Patent Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Patent Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0053] Therefore, as will be apparent to those skilled in the art, the present invention also provides: fragments of F(ab')2Fab, Fv, and Fd; chimeric antibodies, in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies, in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies, in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies, in which the FR and / or CDR1 and / or CDR2 regions are replaced by homologous human or non-human sequences. The present invention also includes so-called single-chain antibodies.

[0054] Various antibody molecules and fragments can be derived from any of the well-known immunoglobulin classes, which include, but are not limited to, IgA, secretory IgA, IgE, IgG, and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. In preferred embodiments, the compound of the present invention is human IgG4.

[0055] In another embodiment, the antibody according to the present invention is a single-domain antibody. The terms “single-domain antibody” (sdAb) or “VHH” refer to a type of antibody found in camelid mammals, which naturally lacks a light chain. Such VHHs are also referred to as “nanobody®”. According to the present invention, the sdAb can be, in particular, a llama sdAb. The term “VHH” refers to a single heavy chain having three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. The terms “complementarity-determining region” or “CDR” refer to a hypervariable amino acid sequence that defines the binding affinity and specificity of the VHH.

[0056] The VHH according to the present invention can be readily prepared by those skilled in the art using routine experiments. VHH variants and their modifications can be produced by any technique known in the art, for example, under in vitro maturation.

[0057] VHHs or sdAbs are typically produced by PCR cloning a V-domain repertoire derived from cDNA of blood, lymph nodes, or spleen obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are usually selected by panning a phage library onto immobilized antigens, for example, antigens coated on the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often exhibit lower affinity for their antigens than VHHs derived from animals that have undergone multiple immunizations. The high affinity of VHHs from immunization libraries is attributed to the natural selection of variant VHHs during the clonal proliferation process of B cells in the lymphoid organs of immunized animals. The affinity of VHH from non-immune libraries can often be improved by mimicking such strategies in vitro, namely by site-directed mutagenesis of the CDR region and further rounds of panning on immobilized antigens under enhanced stringency conditions (higher temperature, higher or lower salt concentrations, higher or lower pH, and lower antigen concentrations). Camelid-derived VHH are readily expressed in E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies and then purified. VHH generally exhibit high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, "Hamers' patents" describe methods and techniques for producing VHH against any desired target (see, e.g., U.S. Patent No. 5,800,988; U.S. Patent No. 5,874,541 and U.S. Patent No. 6,015,695).The "Hamers Patent" specifically describes the production of VHH in bacterial hosts, such as Escherichia coli (see, for example, U.S. Patent No. 6,765,087), and in lower eukaryotic hosts, such as fungi (e.g., Aspergillus or Trichoderma), or in yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see, for example, U.S. Patent No. 6,838,254).

[0058] In another aspect, the present invention provides an antibody that competes with the antibody of the present invention for binding to a target.

[0059] As used herein, the term “binding” in the context of antibody binding to a given antigen or epitope typically refers to binding with an affinity corresponding to a KD of approximately 10⁻⁷ M or less, e.g., approximately 10⁻⁸ M or less, e.g., approximately 10⁻⁹ M or less, approximately 10⁻¹⁰ M or less, or even approximately 10⁻¹¹ M or less, as determined by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument, using the antigen in soluble form as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats routinely used in bin panels of monoclonal antibody epitopes. Normally, an antibody binds to a given antigen that has an affinity equivalent to its KD (Key Derivedness) of a nonspecific antigen (e.g., BSA, casein), such that the KD is less than one-tenth, for example, less than one-hundredth, for example, less than one-thousandth, for example, less than one-tenth,000th, for example, one-hundred-thousandth. A nonspecific antigen is either not identical to or not closely related to the given antigen. If the antibody's KD is very low (i.e., the antibody has high affinity), then the KD to which the antibody binds to the antigen is usually less than one-tenth,000th of its KD for the nonspecific antigen. If such binding is undetectable (for example, using plasmon resonance (SPR) technology in a BIAcore 3000 instrument with a soluble antigen as the ligand and an antibody as the analyte), or if such detection is less than 1 / 100, 1 / 500, or 1 / 1000 of the binding detected by antigens or epitopes having a different chemical structure or amino acid sequence from the antibody, then the antibody is said not to essentially bind to the antigen or epitope.

[0060] Further antibodies can be identified based on their ability to cross-compete with other antibodies of the present invention in a standard antigen-binding assay (e.g., competitively inhibiting the binding of other antibodies of the present invention in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody of the present invention to a target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to a non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitopes of targets as the antibody it competes with. Thus, another aspect of the present invention provides an antibody that binds to the same antigen as the antibody disclosed herein and competes with it. As used herein, an antibody "competes" for binding if, in the presence of equimolar concentrations of the competing antibody, it inhibits the target binding of the antibody or antigen-binding fragment of the present invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0061] In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of a target. In some embodiments, the epitope to which the antibody or antigen-binding fragment of the present invention binds is a linear epitope. In other embodiments, the epitope to which the antibody or antigen-binding fragment of the present invention binds is a non-linear three-dimensional epitope.

[0062] In one embodiment, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is a CD44 expression inhibitor.

[0063] When used in the context of gene or nucleic acid expression, the term “expression” refers to the conversion of information contained in a gene into a gene product. A gene product may be the direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by the translation of mRNA. Gene products also include messenger RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristylation, and glycosylation.

[0064] "Expression inhibitor" refers to a natural or synthetic compound that has a biological effect of inhibiting gene expression. "Expression inhibitor" refers to any compound that has a biological effect of inhibiting the expression of a target gene and / or a target protein. In one embodiment of the present invention, the expression inhibitor is short hairpin RNA (shRNA), small inhibitory RNA (siRNA), or antisense oligonucleotide. Preferably, the expression inhibitor is siRNA or shRNA.

[0065] Target expression inhibitors for use in the present invention can be based on antisense oligonucleotide constructs. Antisense oligonucleotides, which include antisense RNA molecules and antisense DNA molecules, are thought to act as follows: by binding to target mRNA, they directly block the translation of target mRNA, thus inhibiting protein translation or increasing mRNA degradation, thereby reducing the level of the target protein in the cell and consequently reducing its activity. For example, antisense oligonucleotides of at least about 15 nucleotides and complementary to a unique region of the mRNA transcription sequence encoding the target can be synthesized, for example, by conventional phosphodiester techniques and administered, for example, by intravenous injection or infusion. Methods using antisense techniques to specifically reduce the gene expression of a gene whose sequence is known are known in the art (see, for example, U.S. Patents 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0066] Small inhibitory RNAs (siRNAs) can also function as inhibitors of target expression for use in the present invention. Target gene expression can be reduced by contacting the target or cells with small double-stranded RNA (dsRNA) or a vector or construct that induces the production of small double-stranded RNA, resulting in specific inhibition of target expression (i.e., RNA interference or RNAi). For genes whose sequences are known, methods for selecting a suitable dsRNA or dsRNA-encoding vector are well known in the art (see, for example, Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; and International Publication Nos. 01 / 36646, 99 / 32619 and 01 / 68836).

[0067] Short hairpin RNA (shRNA) or small inhibitory RNA (siRNA) can function as gene expression inhibitors for use in the present invention. Gene expression can be reduced using small double-stranded RNA (dsRNA), or vectors or constructs that induce the generation of small double-stranded RNA, resulting in specific inhibition of gene expression (i.e., RNA interference or RNAi). For genes whose sequences are known, methods for selecting appropriate dsRNA or dsRNA-encoding vectors are well known in the art.

[0068] Ribozymes can also function as target expression inhibitors in the present invention. Ribozymes are enzyme RNA molecules that can catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonuclease cleavage. Manipulated hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonuclease cleavage of target mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are typically identified by scanning the target molecule for ribozyme cleavage sites including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural properties, such as secondary structures that may impair the oligonucleotide sequence. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, for example, using a ribonuclease protection assay.

[0069] Both antisense oligonucleotides (ODNs) and ribozymes, which are useful as target inhibitors, can be prepared by known methods. These include chemical synthesis techniques, such as solid-phase phosphoramidite chemosynthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of a DNA sequence encoding an RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors incorporating a suitable RNA polymerase promoter, for example, a T7 or SP6 polymerase promoter. Various modifications can be introduced into the oligonucleotides of the present invention as a means of improving intracellular stability and extending half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'-O-methyl molecules rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0070] The antisense oligonucleotides, siRNAs, and ribozymes of the present invention can be delivered in vivo, either alone or in association with a vector. In its broadest sense, “vector” is any vehicle that can facilitate the transfer of an antisense oligonucleotide, siRNA, or ribozyme nucleic acid into a cell, preferably a cell expressing the target. Preferably, the vector transports the nucleic acid into the cell with reduced degradation compared to the degree of degradation that would occur in the absence of the vector. Overall, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, viral sources, or other vehicles derived from bacterial sources that have been manipulated by the insertion or incorporation of an antisense oligonucleotide, siRNA, or ribozyme nucleic acid sequence. Viral vectors are preferred types of vectors, and these include, but are not limited to, nucleic acid sequences derived from viruses such as: retroviruses, e.g., Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses, e.g., retroviruses. Other vectors known in the art but not explicitly named can readily be used.

[0071] Preferred viral vectors are those based on non-cellularly invasive eukaryotic viruses, in which non-essential genes are replaced with the target gene. Examples of non-cellularly invasive viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral integration into host cell DNA. Retroviruses have been approved in human gene therapy trials. Most useful are retroviruses with replication defects (i.e., those that can direct the synthesis of the desired protein but cannot produce infectious particles). Such genetically modified retroviral expression vectors have general applications for highly efficient gene transduction in vivo. Standard protocols for generating replication-deficient retroviruses (which include steps such as incorporating exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retrovirus by the packaging cell line, collection of viral particles from tissue culture medium, and infection of target cells with the viral particles) are provided in KRIEGLER ("A Laboratory Manual," WH Freeman CO, New York, 1990) and MURRY ("Methods in Molecular Biology," Vol. 7, Humana Press, Inc., Cliffton, NJ, 1991).

[0072] Viruses preferred for certain applications are adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses already approved for human use in gene therapy. Adeno-associated viruses can be manipulated to be replication-deficient and can infect a wide range of cell types and species. This has further advantages, such as thermal and lipid solvent stability; high transduction frequency in diverse cell lineages, including hematopoietic cells; and lack of inhibition of co-infection, which allows for transduction of multiple lineages. It has been reported that adeno-associated viruses can integrate into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in inserted gene expression characteristic of retroviral infections. In addition, wild-type adeno-associated virus infection was tracked in tissue culture for more than 100 passages without selective pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0073] Other vectors include plasmid vectors. Plasmid vectors are extensively described in the art and are well known to those skilled in the art. See, for example, SANBROOK et al., "Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been used as DNA vaccines for delivering antigen-coding genes to cells in vivo. Plasmid vectors have a particular advantage in this respect, as they do not have the same safety concerns as many viral vectors. However, these plasmids have promoters that are compatible with host cells and can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScrip. Other plasmids are well known to those skilled in the art. In addition, plasmids can be custom designed using restriction enzyme and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by a variety of parenteral, mucosal, and local routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or via other routes. DNA plasmids can also be administered as intranasal sprays or drops, rectal suppositories, and orally. They can also be administered to the epidermal or mucosal surface using a gene gun. Plasmids can be delivered in aqueous solution, dried onto gold particles, or in association with other DNA delivery systems, including but not limited to liposomes, dendrimers, cocreates, and microencapsulation.

[0074] In some embodiments, the CD44 expression inhibitor is a nucleic acid that inhibits the CD44 gene, as described in Chinese Patent No. 104561000.

[0075] In another aspect of this disclosure, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is an iron chelating agent.

[0076] As used herein, the term “iron chelator” has its general meaning in the art and refers to any compound that chelates iron and subsequently depletes iron at the cellular level. The term “iron chelator” also refers to compounds that remove excess or toxic iron from a patient’s blood, tissues, and organs.

[0077] Iron chelating agents are well known in the art, for example, as described below: International Publication No. 0149266; International Publication No. 2009103727; International Publication No. 2011109521; International Publication No. 2014059417; International Publication No. 2017153475; International Publication No. 2019233982 and Lima et al., Treatment of iron overload syndrome: a general review. Rev Assoc Med Bras (1992). October 10, 2019; 65(9): pp. 1216-1222. doi:10.1590 / 1806-9282.65.9.1216; Mobarra et al., A Review on Iron Chelators in Treatment of Iron Overload Syndromes. Int J Hematol Oncol Stem Cell Res. October 1, 2016; 10(4): pp. 239-247; Khan et al., Synthesis, nature and utility of universal iron chelator Siderophore: A review. Microbiol Res. 2018 July-August;212-213: pp. 103-111. doi: 10.1016 / j.micres.2017.10.012; Khan et al., Synthesis, nature and utility of universal iron chelator Siderophore: A review. Microbiol Res. July-August 2018;212-213: pp. 103-111. doi: 10.1016 / j.micres.2017.10.012; Kontoghiorghes GJ. Comparative efficacy and toxicity of desferrioxamine, deferiprone and other iron and aluminum chelating drugs. Toxicol Lett. October 1995;80(1-3):pp.1-18.

[0078] Tests and assays for determining whether a compound is an iron chelating agent are well known to those skilled in the art, as described below: International Publication No. 0149266; International Publication No. 2009103727; International Publication No. 2011109521; International Publication No. 2014059417; International Publication No. 2017153475; International Publication No. 2019233982.

[0079] In some embodiments, iron chelating agents are selected from the group consisting of, but are not limited to, the following: deferoxamine (DFO) (also known as desferral, desferrioxamine, DFB, CAS number: 70-51-9); deferoxamine mesylate (CAS number: 138-14-7); deferoxamine hydrochloride (CAS number: 1950-39-6); deferasirox (DFX); deferipron (DFP); deferritrin; deferipon; metformin; metformin; deferritazole; 3-hydroxy-4-pyridinone; 3-hydroxy-4-pyridinone derivatives; hepcidine and hepcidine fragments (minihepcidine); polyamine-dihydroxybenzoic acid and polyamine-dihydroxybenzoic acid Conjugates of benzoic acid; pyridoxal isonicotinoyl hydrazone (PIH); rhodotol phosphate; N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED), N,N'-bis(2-hydroxybenzyl)propylene-1,3-diamine-N,N-diacetic acid (HBPD), 2,3-dihydroxybenzoic acid; diethyltriaminepentaacetic acid (DTPA); and the compounds listed below: U.S. Patent No. 5,374,771; International Publication No. 0149266; International Publication No. 2009103727; International Publication No. 2011109521; International Publication No. 2014059417; International Publication No. 2017153475; International Publication No. 2019233982; and Lima et al., Treatment of iron overload syndrome: a general review. Rev Assoc Med Bras (1992). 10 Oct 2019 10;65(9):1216~1222. doi:10.1590 / 1806-9282.65.9.1216; Kontoghiorghes GJ. Comparative efficacy and toxicity of desferrioxamine, deferiprone and other iron and aluminum chelating drugs. Toxicol Lett. October 1995;80(1-3):pp.1-18.

[0080] In some embodiments, the iron chelating agent is a derivative of a biguanide, selected from the group consisting of metformin, metformin derivatives such as metformin, and compounds containing the biguanidyl radical described in International Publication No. 2019233982, but is not limited to these.

[0081] In some embodiments, the iron chelating agent is selected from the group consisting of, but is not limited to, bacterial siderophores and siderophores (catecholate, hydroxylate, carboxylate, or mixed types), such as desferrioxamine; catecholate siderophores: enterobactin; vibrioferin; mixed siderophores: yersinia bactin; and phytosiderophores: mugineic acid; and the iron chelating agents described in (Khan et al., Synthesis, nature and utility of universal iron chelator - Siderophore: A review. Microbiol Res. July-August 2018; 212-213: pp. 103-111. doi: 10.1016 / j.micres.2017.10.012).

[0082] In some embodiments, the iron chelating agent is lysosomal iron sequestration low molecular weight ironomycin.

[0083] In a further embodiment, the present invention relates to a method for treating cytokine release syndrome (CRS) in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of a CD44 / hyaluronic acid (HA) pathway antagonist to the subject.

[0084] In some embodiments, the present invention relates to a method for treating severe COVID-19-associated CRS in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of a CD44 / hyaluronic acid (HA) pathway antagonist to the subject.

[0085] In a further embodiment, the present invention relates to a method for treating respiratory distress syndrome (ARDS), macrophage activation syndrome (MAS), iron-related inflammatory disease and / or alveolar inflammatory response in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of a CD44 / hyaluronic acid (HA) pathway antagonist to the subject.

[0086] In a further embodiment, the present invention relates to a method for treating COVID-19, particularly severe COVID-19, in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of a CD44 / hyaluronic acid (HA) pathway antagonist to the subject.

[0087] In some embodiments, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is administered in combination with the treatment of anti-cytokine release syndrome (CRS).

[0088] In some embodiments, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is administered in combination with anti-severe COVID-19-associated CRS treatment.

[0089] In some embodiments, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is administered in combination with any further optional compounds used in the treatment of respiratory distress syndrome (ARDS), macrophage activation syndrome (MAS), iron-related inflammatory diseases and / or alveolar inflammatory reactions.

[0090] In some embodiments, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention is administered in combination with anti-COVID-19 treatment, particularly anti-severe COVID-19 treatment.

[0091] In particular, the CD44 / hyaluronic acid (HA) pathway antagonist of the present invention can be administered in combination with an anti-inflammatory agent.

[0092] The term “treatment for anti-cytokine release syndrome (CRS)” has its general meaning in this art and refers to the treatment of CRS administered to CRS patients and any compound used in any type of CRS therapy, including any compound used in the treatment of CRS (Shimabukuro-Vornhagen et al., Cytokine release syndrome. J Immunother Cancer. June 15, 2018; 6(1): 56. doi: 10.1186 / s40425-018-0343-9; Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science. May 1, 2020; 368(6490): 473-474. doi: 10.1126 / science. abb8925).

[0093] Compounds and therapies used in the treatment of CRS include, but are not limited to, antihistamines; antipyretics; oral or intravenous fluids used in hydration therapy; antibiotic therapy; BiTE blinatumomab; IL-6 antagonists, such as IL-6 targeting agents like siltuximab or IL-6R targeting agents like tocilizumab and sarilumab; anti-TNFα agents, such as etanercept; anti-GM-CSF agents, such as lenzirumab; ibrutinib; and T-cell depletion antibody therapies, such as alemtuzumab and anti-thymocyte globulin (ATG), IL-1R-based inhibitors (anakinra) and cyclophosphamide; and anti-inflammatory agents.

[0094] The term "anti-COVID-19 treatment" has a general meaning in this art and refers to any type of treatment for COVID-19 administered to a person with COVID-19, including any compounds used in the treatment of COVID-19, such as antiviral agents, compounds used in the treatment of severe COVID-19-associated CRS, and anti-inflammatory agents (Zhou et al., M, Zhang X, Qu J. Coronavirus disease 2019 (COVID-19): a clinical update. Front Med. April 2020;14(2): pp. 126-135. doi: 10.1007 / s11684-020-0767-8).

[0095] Compounds and therapies used in the treatment of COVID-19 include, but are not limited to, the following: antiviral agents, e.g., remdesivir (GS-5734), GS-441524, lopinavir, ritonavir, lopinavir / ritonavir (LPV / r) combination, lopinavir / ritonavir and interferon beta-1-alpha combination (LPV / r / INF-β-1a combination), ribavirin, ribavirin and interferon-α combination (ribavirin and INF-α2b combination), and avidol (umifenovir or arbidol); IL-6 antagonists, e.g., IL-6 targeting agents such as siltuximab. Agents that target IL6R, such as tocilizumab and sarilumab; IL-1 antagonists, such as IL-1β anakinra antagonists; TNF blockers; IFN-αβ inhibitors; chloroquine; hydroxychloroquine (HCQ); corticosteroids; antimicrobial agents, such as antibiotics and antifungals; intravenous immunoglobulin (IVIG); convalescent plasma therapy; anticoagulants; oxygen therapy, such as high-flow nasal oxygen therapy (HFNC), non-invasive ventilation (NIV), invasive ventilation and extracorporeal membrane oxygenation (ECMO); continuous renal replacement therapy (CRRT); and compounds used in the treatment of CRS.

[0096] Anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ibuprofen, salicylic acid acetate, sulindac, piroxicam, and naproxen; as well as corticosteroids.

[0097] Pharmaceutical composition The compounds of the present invention can be used or prepared in pharmaceutical compositions.

[0098] In one embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier for use in the treatment of cytokine release syndrome (CRS) in subjects requiring such use.

[0099] In some embodiments, the present invention relates to a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier for use in the treatment of severe COVID-19-related CRS in subjects requiring such use.

[0100] Typically, the compounds of the present invention can be combined with pharmaceutically acceptable excipients and, optionally, with a sustained-release matrix, such as a biodegradable polymer, to form therapeutic compositions.

[0101] Typically, the compounds according to the present invention described above are administered to the patient in a therapeutically effective dose.

[0102] The "therapeutic effective dose" of the compound of the present invention described above indicates a sufficient amount of the compound with a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total daily dose of the compound and composition of the present invention will be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutic effective dose level for any particular patient depends on a variety of factors, including: the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the patient's age, weight, general health, sex, and diet; the timing of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or simultaneously with the specific compound used; and similar factors well known in the field of medical technology. For example, starting the dose of the compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved is well within the scope of the art. However, the daily dose of the product can vary widely from 0.01 to 1,000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the compound of the present invention for symptomatic adjustment of the dosage to the patient being treated. The pharmacopoeia typically contains about 0.01 mg to about 500 mg of the compound of the present invention, preferably 1 mg to about 100 mg of the compound of the present invention. The effective dose of the drug is usually supplied at dosage levels ranging from 0.0002 mg to about 20 mg per kg of body weight per day, and particularly from about 0.001 mg to 7 mg per kg of body weight per day.

[0103] In certain embodiments, the compounds according to the present invention can be used at concentrations between 0.01 μM and 20 μM, and in particular, the compounds of the present invention can be used at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, and 20.0 μM.

[0104] According to the present invention, the compounds of the present invention are administered to a target in the form of a pharmaceutical composition. Typically, the compounds of the present invention can be combined with pharmaceutically acceptable excipients and, optionally, with a sustained-release matrix, such as a biodegradable polymer, to form a therapeutic composition. "pharmaceutically" or "pharmaceutically acceptable" means molecular entities and compositions that, as appropriate, do not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation aid.

[0105] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient may be administered to animals and humans alone or in combination with other active ingredients in unit dosage forms or as mixtures with conventional pharmaceutical supports. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or liquids; sublingual and buccal administration forms; aerosols, implants; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and nasal administration forms; and rectal administration forms.

[0106] Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for an injectable formulation. These can be, in particular, isotonic and sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or, optionally, dry, especially lyophilized, compositions to which sterile water or physiological saline can be added to form an injectable solution. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to have easy syringeability. The form must be stable under manufacturing and storage conditions and must be resistant to microbial contamination, such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmaceutically acceptable salts can be prepared in water appropriately mixed with a surfactant, such as hydroxypropyl cellulose. The dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives that prevent microbial growth. The compounds of the present invention can be formulated into compositions in neutral or salt form. Medicinally acceptable salts include acid addition salts (formed using free amino groups of proteins), which are formed using inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed using free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.Appropriate fluidity can be maintained, for example, by the use of a coating agent, such as lecithin; by maintaining the required particle size in the case of a dispersion; and by the use of a surfactant. Protection against microbial action can be provided by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Extending the absorption of the injectable composition can be achieved by using an absorption-delaying agent in the composition, such as aluminum monostearate and gelatin. A sterile injection solution is prepared by incorporating the required amount of the active compound in a suitable solvent, along with some of the other components listed above if necessary, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterile agents of the present invention into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, typical preparation methods involve vacuum drying and freeze-drying techniques, which yield a powder containing the compound of the present invention plus any additional desired components from a pre-sterilized filtered solution. Preparation of more concentrated or highly concentrated solutions for direct injection is also being considered, in which case the use of DMSO as a solvent is assumed to result in extremely rapid penetration, thereby delivering a high concentration of the active agent to a small tumor area. During formulation, the solution is administered in a manner compatible with the drug formulation and in a therapeutically effective amount. The formulation is readily administered in various dosage forms, such as the injectable solution types described above, but drug-release capsules and the like can also be used. For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be appropriately isotonic using sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in view of this disclosure. Some variation in medication dosage will inevitably occur depending on the condition of the patient being treated.The person responsible for administering the medication will determine the appropriate dose for each individual patient.

[0107] The pharmaceutical composition of the present invention may contain any further compounds used in the treatment of cytokine release syndrome (CRS).

[0108] In some embodiments, the pharmaceutical compositions of the present invention may include any further compounds used in the treatment of severe COVID-19-related CRS.

[0109] In some embodiments, the pharmaceutical compositions of the present invention may include any further compounds used in the treatment of respiratory distress syndrome (ARDS), macrophage activation syndrome (MAS), iron-related inflammatory diseases and / or alveolar inflammatory reactions.

[0110] In some embodiments, the pharmaceutical compositions of the present invention may include any further compounds used in the treatment of COVID-19, particularly severe COVID-19.

[0111] In one embodiment, the further active compounds may be contained in the same composition or administered separately.

[0112] In another embodiment, the pharmaceutical composition of the present invention relates to combination preparations for simultaneous, separate, or sequential use in the treatment of cytokine release syndrome (CRS) in subjects requiring it.

[0113] In some embodiments, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of severe COVID-19-related CRS in subjects requiring such treatment.

[0114] In another embodiment, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of respiratory distress syndrome (ARDS), macrophage activation syndrome (MAS), iron-related inflammatory diseases and / or alveolar inflammatory reactions.

[0115] In another embodiment, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of COVID-19, particularly severe COVID-19.

[0116] The present invention also provides a kit containing the compound of the present invention. The kit containing the compound of the present invention has potential applications in therapeutic methods.

[0117] Diagnostic methods In a further aspect, the present invention relates to using CD44 as a biomarker of predisposition to severe COVID-19, and then predicting the occurrence of severe COVID-19.

[0118] Accordingly, the present invention relates to a method for identifying a subject who has severe COVID-19, or is at risk of having or developing it, the method comprising the step of measuring the expression level of CD44 in a biological sample obtained from said subject.

[0119] As used herein, the term “biological sample” means any biological sample derived from the subject, such as a blood sample, peripheral blood sample, bronchoalveolar sample, bronchial sample, alveolar sample, nasopharyngeal sample, sputum sample, mucus sample, tracheal sample, or nasal cavity sample.

[0120] The method of the present invention may further include a step of comparing the expression level of CD44 in a biological sample with a reference value, wherein detecting a difference in the expression level of CD44 between the biological sample and the reference value indicates a subject who has or is at risk of having or developing severe COVID-19.

[0121] As used herein, “reference value” refers to a threshold or cutoff value. Typically, a “threshold” or “cutoff value” can be determined experimentally, empirically, or theoretically. Thresholds can also be arbitrarily selected based on existing experimental and / or clinical conditions, as will be understood by those skilled in the art. Thresholds are required to be determined to obtain optimal sensitivity and specificity according to the function of the test and the balance of benefit / risk (clinical outcomes of false positives and false negatives). Typically, optimal sensitivity and specificity (and thus thresholds) can be determined using receiver operating characteristic (ROC) curves based on experimental data. Preferably, those skilled in the art can compare the expression level (obtained according to the method of the present invention) to a specified threshold. In one embodiment of the present invention, the threshold is derived from the expression level (or ratio, or score) determined in biological samples from one or more subjects with severe COVID-19. Furthermore, retrospective measurements of expression levels (or ratio, or score) in appropriately deposited historical subject samples can be used to establish these thresholds.

[0122] In one embodiment, the reference value may correspond to the CD44 expression level determined in a biological sample associated with a subject who does not have severe COVID-19, or who has it or is not at risk of developing it. Therefore, a CD44 expression level higher than the reference value indicates a subject who has severe COVID-19, or who has it or is at risk of developing it, while a CD44 expression level lower than or equal to the reference value indicates a subject who does not have severe COVID-19, or who has it or is not at risk of developing it.

[0123] In another embodiment, the reference value may correspond to the CD44 expression level determined in a biological sample associated with a subject having severe COVID-19 or at risk of having or developing it. Thus, a CD44 expression level higher than or equal to the reference value indicates a subject having severe COVID-19 or at risk of having or developing it, while a CD44 expression level lower than the reference value indicates a subject not having severe COVID-19 or at risk of having or developing it.

[0124] CD44 expression levels can be evaluated using one of a wide variety of well-known methods for detecting the expression of transcribed nucleic acids or translated proteins.

[0125] In one embodiment, the expression level of CD44 is evaluated by analyzing the expression of the mRNA transcript or mRNA precursor of the CD44 gene, for example, nascent RNA. This analysis can be performed by preparing mRNA / cDNA from cells of a biological sample from the subject and hybridizing the mRNA / cDNA with a reference polynucleotide. The prepared mRNA / cDNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern DNA analysis, polymerase chain reaction analysis, e.g., quantitative PCR (TaqMan), and probe arrays, e.g., GeneChip™ DNA Arrays (AFFYMETRIX).

[0126] Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding CD44 involves a nucleic acid amplification process, such as RT-PCR (experimental embodiment described in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991), auto-persistent sequence replication (Guatelli et al., 1990), transcription amplification system (Kwoh et al., 1989), Q-beta replicase (Lizardi et al., 1988), rolling circle replication (U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when they are present in very small numbers. As used herein, amplification primers are defined as a pair of nucleic acid molecules that can anneal to the 5' or 3' region of a gene (the positive and negative strands, respectively, or vice versa) and may contain a short region in between. Generally, amplification primers are approximately 10 to 30 nucleotides long and adjacency to regions of approximately 50 to 200 nucleotides long. Under appropriate conditions and with appropriate reagents, such primers can amplify nucleic acid molecules containing the nucleotide sequences adjacent to the primers.

[0127] In another embodiment, the expression level of CD44 is assessed by analyzing the expression of the protein translated from the gene. This analysis can be performed using antibodies (e.g., radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibodies), antibody derivatives (e.g., antibody conjugates with a substrate or a portion of a protein / ligand pair protein or ligand (e.g., biotin-streptavidin)), or antibody fragments (e.g., single-chain antibodies, isolated antibody hypervariable domains, etc.), which specifically bind to the protein translated from the gene encoding CD44.

[0128] The aforementioned analysis can be evaluated by various techniques well known to those skilled in the art, including, but not limited to, enzyme immunoassays (EIA), radioimmunoassays (RIA), Western blot analysis, and enzyme-linked immunosorbent assays (RIA).

[0129] In a further embodiment, the method of the present invention is carried out by identifying macrophage activation.

[0130] In one embodiment, macrophage activation is evaluated by measuring the cytokine profile. Analysis of macrophage activation can be evaluated according to the method of the present invention.

[0131] A further aspect of the present invention relates to a method for monitoring the progression of COVID-19 by carrying out the method of the present invention.

[0132] In one embodiment, the present invention relates to a method for treating severe COVID-19 in a person requiring it, comprising the following steps: (i) A step of identifying a subject who has severe COVID-19 or is at risk of having or developing it by carrying out the method according to the present invention, (ii) If it is determined that the subject has severe COVID-19 or is at risk of having or developing it, the step of administering a CD44 / hyaluronic acid (HA) pathway antagonist to the subject.

[0133] In a further embodiment, the present invention relates to using CD44 as a biomarker for the efficiency of the compound of the present invention, and then predicting the subject's response to the compound of the present invention.

[0134] Accordingly, the present invention relates to a method for determining whether a subject suffering from cytokine release syndrome (CRS) achieves a response to an antagonist of the CD44 / hyaluronic acid (HA) pathway, comprising the step of determining the expression level of CD44, wherein the expression level is related to the subject's response to treatment.

[0135] In another aspect, the present invention relates to a method for determining whether a subject suffering from COVID-19, particularly severe COVID-19, achieves a response to an antagonist of the CD44 / hyaluronic acid (HA) pathway, comprising the step of determining the expression level of CD44, wherein the expression level is related to the subject's response to treatment.

[0136] In some embodiments, the method includes the steps of: i) determining the expression level of CD44; ii) comparing the expression level determined in step i) with a predetermined reference value; and iii) concluding that the subject achieves a response if the level determined in step i) is higher than the predetermined reference value.

[0137] Therefore, the method is particularly suitable for distinguishing responders from non-responders. As used herein, the term “responder” in the context of this disclosure refers to a subject that achieves a response, i.e., a subject whose disease is eradicated, mitigated, or improved. According to the present invention, a responder has an objective response, and therefore the term does not include subjects with a stabilized disease, so that the disease has not progressed after treatment. Non-responders or unresponsive subjects include subjects whose disease does not show mitigation or improvement after treatment. According to the present invention, the term “non-responder” also includes subjects with a stabilized disease. Typically, characterizing subjects as responders or non-responders can be done by referring to criteria or training sets. The criteria may be profiles of subjects known to be responders or non-responders, or they may be numerical. Such predetermined criteria may be provided in any suitable form, for example, in printed lists or diagrams, computer software programs, or other media. If a patient is concluded to be a non-responder, the physician may decide to discontinue and modify treatment to avoid any further adverse side effects.

[0138] In one embodiment, the present invention relates to a method for treating severe COVID-19 in a person requiring it, comprising the following steps: (i) A step of determining whether the subject achieves a response by an antagonist of the CD44 / hyaluronic acid (HA) pathway by carrying out the method according to the present invention, (ii) If the subject is concluded to be a responder, the step of administering a CD44 / hyaluronic acid (HA) pathway antagonist to the subject, This includes methods.

[0139] The present invention is further illustrated by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of the present invention in any way. figure [Brief explanation of the drawing]

[0140] [Figure 1] This figure shows the increase in CD44 and RhoNOX-M in activated macrophages. GM-CSF-treated monocytes (M1) were activated with LPS (100 ng / mL) for 24 hours, and CSF1-treated monocytes were activated with IL4 (20 ng / mL) for 24 hours. CD44, TfR, and RhoNOX-M were measured by flow cytometry. [Figure 2] This is a diagram illustrating CD44-dependent iron endocytosis during macrophage activation. [Figure 3] This figure shows that CD44 antagonists induce a decrease in intracellular iron levels in activated monocyte-derived macrophages. (A) ICP-MS of intracellular iron and copper in activated (act.) monocyte-derived macrophages (MDM) treated with siCtrl. or siCD44. n=7 donors. (B) Representative Western blot of CD44 in act. MDM transfected with siRNA against CD44 or control siRNA. n=4 donors. Mann-Whitney test. Mean ± SEM. (C) ICP-MS of intracellular iron and copper in act. MDM treated with anti-CD44 antibody RG7356. n=7 donors. Different donors are shown separately as dots in each panel. [Examples]

[0141] (Example 1) Materials and methods Chemical reagents and antibodies. GM-CSF and IFN-gamma were obtained from Miltenyi (Miltenyi Biotec, Somerville, MA, USA), deferoxamine (DFO) from Sigma-Aldrich (Saint-Quentin Fallavier, France), RhoNox-M (in-house), Hyaluronate Fluorescein (FITC-Hyal) (#YH4532, Carbosynth, San Diego, CA, USA), and human serum-derived transferrin and Alexa Fluor 647 conjugate (TF-647) (#T23366) from ThermoFisher Scientific (Waltham, MA, USA). Antibodies against CD44 (#ab189524, WB: 1:30000, Abcam, Cambridge, MA, USA), ferritin (#ab75973, WB: 1:1000, Abcam), H3K9me2 (#4658S, WB: 1:1000, Cell Signaling, Danvers, MA, USA), and transferrin receptor 1 (TfR1) (#13-6800, WB: 1:1000, Thermo Fisher Scientific) were used.

[0142] Cell culture. Peripheral blood samples were collected from healthy donors. Panmonocytes were sorted using microbeads according to the manufacturer's instructions (Miltenyi Biotec, Somerville, MA, USA), cultured in RPMI 1640 containing glutamine (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, and exposed to GM-CSF 100 ng / mL to induce differentiation. After 5 days of differentiation, macrophages were activated with IFN-g (50 ng / mL) and treated with DF0 (10 μm) for 24 hours.

[0143] Bronchoalveolar lavage (BAL). Alveolar macrophages were purified by centrifuging BAL from COVID patients and then adhering it to plastic dishes of serum or antibiotic-free serum RPMI 1640 medium at 37°C in a humidified atmosphere containing 5% CO2. After incubation for approximately 15-30 minutes, non-adherent cells were removed, and the layer of adherent cells was washed twice with 10 mL of ice-cold sterile PBS. Adherent cells were treated overnight with DFO or RG7356.

[0144] Cell morphology. Phase-contrast images were captured using a CKX41 microscope (Olympus) and cellSens Entry imaging software (Olympus). Subsequently, the long and short axes (long axis: the longest length of the cell; short axis: the length across the nucleus perpendicular to the long axis) of each cell were manually traced, and the elongation factor was measured as the ratio of these axes.

[0145] Flow cytometry analysis of cellular phenotypes. Cells were washed with ice-cold PBS, incubated with Fc blocks (Human TruStain FcX, Biolegend, London, UK, 1 / 20) for 15 minutes, incubated with antibodies at 4°C for 20 minutes, washed, and then analyzed using BD LSRFortessa X-20. The antibodies used were AlexaFluor700-CD80 (#561133, BD, Franklin Lakes, NJ, USA), PE / Cy7-CD86 (#561128, BD), APC / Alexa750-CD71 (#A89313, Beckman Coulter, Brea, CA, USA), Krome orange-CD14 (#B01175, Beckman Coulter), Pacific Blue-CD16 (#A82792, Beckman Coulter), PE-CD163 (#556018, BD), and AlexaFluor647-CD44 (#NB500-481AF647, Novus Biologicals). The data were analyzed using FlowJo software v. 10.0.00003.

[0146] Immunofluorescence microscopy. Cells were spotted onto slides using cytospin centrifugation, washed with PBS, fixed with 4% formaldehyde in PBS for 20 minutes, washed with PBS, post-fixed, permeabilized with cold 70% ethanol for 20 minutes, washed with PBS, blocked with 8% bovine serum albumin (BSA) in PBS for 1 hour, incubated with primary antibody in 1% BSA in PBS for 2 hours, washed, incubated with secondary antibody conjugated with Alexa Fluor 488 or 555 for 1 hour, washed, and mounted using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA).

[0147] Other fluorescence imaging experiments: Live cells were incubated in a medium containing RhoNox-M, FITC-Hyal, and TF-647 at 37°C for 1 hour with 5% CO2. The cells were then washed three times with PBS, spotted onto slides using cytospin centrifugation, and mounted using Vectashield mounting medium (Vector Laboratories) containing DAPI.

[0148] Fluorescence images were acquired using a Deltavision real-time microscope (Applied Precision). 40× / 1.4NA, 60× / 1.4NA, and 100× / 1.4NA objective lenses were used for 2D and 3D acquisition. The data was deconvolved using SoftWorx (Rayshaw Conservative - 15 iterations, Applied Precision) and processed in ImageJ. All images were acquired as z-stacks.

[0149] Inductively coupled plasma mass spectrometry. Glass vials with Teflon septums were cleaned with 65% nitric acid (VWR), washed with ultrapure water (Sigma-Aldrich), and dried. Cells were plated 24 hours prior to the experiment. In all experiments, cells were incubated in FBS-free medium for 2 hours before processing. Cells were harvested and subsequently washed twice with PBS. Cells were then counted using an automated cell counter (Entek), transferred to a clean vial in 100 μL of PBS, and the sample was lyophilized using a lyophilizer (CHRIST). The sample was then mixed with 65% nitric acid overnight, followed by heating at 80°C for 2 hours. The sample was diluted with ultrapure water (Sigma-Aldrich) to a final concentration of 0.475 N nitric acid and transferred to a metal-free centrifuge vial (VWR) for subsequent ICP-MS analysis. An Agilent 7900 ICP-QMS was used in low-resolution mode. 56 Fe concentration was measured. Sample introduction was performed using a micronebulizer (MicroMist, 0.2 mL / min) through a Scott spray chamber. Isotopes were measured using a collision reaction interface with helium gas (5 mL / min) to eliminate polyatomic interference. Scandium and indium internal standards were injected after inline mixing with the sample to control for signal drift and lack of matrix effects. A mixture of certified standards was measured at concentrations spanning the sample concentration to convert count measurements to concentrations in solution. The uncertainty of the sample concentration was calculated using the algebraic carrier and sample count uncertainty of the ICP-MS blank. Values ​​were normalized by dry weight and cell number.

[0150] Immunoblot analysis. Cells were washed twice with PBS, dissolved in 2× Laemmli buffer containing benzonase (#VWR, Fontenay-Sous-Bois, France), and the extract was incubated at 37°C for 1 hour. Quantification was performed using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were separated using Nu-PAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and electroblotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Milk-saturated membranes were incubated overnight with primary antibody at 4°C, followed by incubation with peroxidase-conjugated anti-IgG secondary antibody (Jackson Laboratories, Bar Harbor, ME, USA) for 45 minutes. The signal was made visible by autoradiography using a SuperSignal West Pico PLUS or West Femto-enhanced chemiluminescence detection kit (Thermo Fisher Scientific).

[0151] Cytokine profiles in human macrophage supernatant. IL1-beta, IL4, IL6, IL8, IL10, IL12, IFN-gamma, and TNF-alpha concentrations were measured in cell culture supernatant using the Human Pro-Inflammatory Combo 1 U-Plex (MSD, Rockville, MD, USA). The kit was run according to the manufacturer's guidelines, and the chemiluminescence signal was measured using a Sector Imager 2400 (MSD).

[0152] RNA sequencing was performed. RNA extracted using the RNeasy mini kit (QIAGEN, Germantown, MD, USA) was processed using the SureSelect Automated Strand Specific RNA Library Preparation Kit and sequenced on an Illumina Novaseq 6000 in paired-end 100bp mode to achieve at least 40 million reads per sample. Gene expression was validated by qRT-PCR as listed below.

[0153] result Our previously unpublished data indicate that during the activation of M1 macrophages, iron endocytosis is upregulated in a CD44-dependent manner, and CD44 protein levels increase. Furthermore, as observed by RhoNox-1, lysosomal Fe is elevated in activated M1 macrophages. 2+ This increases. This effect is specific to M1 macrophages, and the levels of the canonical iron endocytosis protein TfR1 / CD71 remain unchanged (Figures 1 and 2).

[0154] (Example 2) Materials and methods Cell culture. Peripheral blood samples were collected from different healthy donors (Etablissement Francais du Sang). Panmonocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), cultured in RPMI 1640 supplemented with glutamine (Thermo Fisher Scientific, 61870010) and 10% fetal bovine serum, and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec, 130-093-866, 100 ng / mL) to induce differentiation into macrophages (MDM). On day 5 of differentiation, MDM was treated with lipopolysaccharide (LPS, InvivoGen, tlrl-3pelps, 100 ng / mL, 24 hours) and interferon-γ (IFNγ, Miltenyi Biotec, 130-096-484, 20 ng / mL, 24 hours) to generate activated MDM (act. MDM), which was then co-treated with the anti-CD44 antibody RG7356 or transfected with a control or CD44 siRNA using the AMAXA Nucleofector transfection system (LONZA). The following small interfering RNAs are suitable for specific downregulation of CD44: 5' -GAAUAUAACCUGCCGCUUU-3'(Sequence ID 1), 5' -CAAGUGGACUCAACGGAGA-3' (Sequence ID 2), 5'-CGAAGAAGGUGUGUGGGCAGA-3' (SEQ ID NO: 3), and 5' -GAUCAACAGUGGCAAUGGA-3' (Sequence ID 4).

[0155] Western blotting. Cells were processed as instructed and then washed with 1×PBS. Proteins were dissolved in 2× Laemmli buffer containing benzonase (VWR, 70664-3, 1:100), and the extracts were incubated at 37°C for 1 hour and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were separated by SDS-PAGE electrophoresis (Invitrogen sure-lock system and Nu-PAGE 4-12% Bis-Tris precast gel) and transferred to nitrocellulose membranes (Amersham Protran 0.45 μm) using a Trans-Blot SD semi-dry electrophoresis transfer cell (Bio-rad). The membranes were blocked for 1 hour with 5% defatted skim milk powder in 0.1% Tween-20 / 1×PBS. Next, the blot was probed overnight at 4°C using slow-movement techniques with the associated primary antibody in 5% BSA and 0.1% Tween-20 / 1×PBS. The membrane was washed three times with 0.1% Tween-20 / 1×PBS and incubated with horseradish peroxidase conjugate secondary antibody (Jackson Laboratories) in 5% defatted skim milk powder and 0.1% Tween-20 / 1×PBS at room temperature for 1 hour, followed by three washes with 0.1% Tween-20 / 1×PBS. The antigen was detected using the SuperSignal West Pico PLUS chemiluminescence detection kit (ThermoFisher Scientific, 34580 and 34096). The signal was recorded using the Fusion Solo S Imaging System (Vilber) and quantified in ImageJ using pixel intensity normalized for the γ-tubulin signal.

[0156] Inductively coupled plasma mass spectrometry (ICP-MS). Cells were treated for 24 hours with HA (Carbosynth, FH45321, 600-1000 kDa, 1 mg / mL) added together with LPS and IFNγ. Glass vials equipped with teflon septums were cleaned with 65% nitric acid (VWR, Suprapur, 1.00441.0250), washed with ultrapure water (Sigma-Aldrich, 1012620500), and dried. Cells were collected and subsequently washed twice with 1×PBS. Cells were then counted using an automated cell counter (Entek), transferred to clean glass vials in 200 μL of 1×PBS, and the samples were lyophilized using a freeze-dryer (CHRIST, 22080). The samples were then mixed with 65% nitric acid overnight and heated at 80°C for 2 hours. The sample was diluted with ultrapure water to a final concentration of 0.475 N nitric acid and transferred to a metal-free centrifuge vial (VWR, 89049-172) for subsequent ICP-MS analysis. Using an Agilent 7900 ICP-QMS in low-resolution mode, 56 Fe and 63 The amount of Cu was measured. Sample introduction was performed using a micronebulizer (MicroMist, 0.2 mL / min) through a Scott spray chamber. Isotopes were measured using a collision reaction interface with helium gas (5 mL / min) to eliminate polyatomic interference. Scandium and indium internal standards were injected after inline mixing with the sample to control for signal drift and lack of matrix effects. A mixture of certified standards was measured at concentrations spanning the sample concentration to convert count measurements to concentrations in solution. The uncertainty of the sample concentration was calculated using the algebraic carrier and sample count uncertainty of the ICP-MS blank. Values ​​were normalized to cell number.

[0157] result Downregulation of CD44 using small interfering RNA (siRNA) against CD44 reduced the total iron content in activated monocyte-derived M1 macrophages (act. MDM) compared to control RNA (Figures 3A and 3B). In addition, treatment with a CD44-binding blocking antibody (RG7356) reduced the total iron content in act MDM (Figure 3C). These data confirm that CD44 mediates iron uptake during the activation process of inflammatory macrophages.

[0158] In summary, these data suggest a direct role for CD44-mediated iron endocytosis in the severe inflammatory response observed in SARS-CoV-2 patients. Institut Curie has extensive experience with the use of RG7356, a clinically approved anti-CD44 antibody, which has shown promising results in cancer patients with poor outcomes (First-in human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget (2016), DOI: 10.18632 / oncotarget.11098). Herein, we evaluate the effects of using CD44 / hyaluronic acid (HA) pathway antagonists, and the effects of targeting iron homeostasis, for example, by antagonizing IL6 signaling using DFO and protecting SARS-CoV-2 patients from CRS.

[0159] References: Throughout this application, the current state of the art to which the present invention belongs is described by various references. The disclosures of these references are incorporated into this disclosure by reference. [Table 1-1] [Table 1-2] Table 1-3

Claims

1. A pharmaceutical composition for use in the treatment of cytokine release syndrome in a subject requiring the use of the CD44 / hyaluronic acid pathway antagonist, The antagonist of the CD44 / hyaluronic acid (HA) pathway is selected from the group consisting of CD44 antagonists and CD44 expression inhibitors, and is selected from the group consisting of compounds that target CD44. The aforementioned CD44 antagonist is an anti-CD44 antibody. A pharmaceutical composition wherein the CD44 expression inhibitor is selected from the group consisting of antisense oligonucleotides, shRNA, siRNA, and RNAi.

2. The pharmaceutical composition according to claim 1, wherein the cytokine release syndrome is severe COVID-19-related cytokine release syndrome.

3. The pharmaceutical composition according to claim 1, wherein the anti-CD44 antibody is selected from the group consisting of RG7356 and vivacuzumab.

4. A pharmaceutical composition according to any one of claims 1 to 3, for use in the treatment of respiratory distress syndrome (ARDS) in subjects requiring it.

5. A pharmaceutical composition according to any one of claims 1 to 3, for use in the treatment of macrophage activation syndrome (MAS) in subjects requiring such treatment.

6. A pharmaceutical composition according to any one of claims 1 to 3, for use in the treatment of iron-related inflammatory diseases and alveolar inflammatory reactions in subjects requiring such treatment.

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