Granulocyte-macrophage colony-stimulating factor-based treatment of infections

Abstract

The present disclosure relates to the treatment of coronavirus infections using granulocyte-macrophage colony-stimulating factor.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 014,462, filed on April 23, 2020, and U.S. Provisional Patent Application No. 63 / 093,576, filed on October 19, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present invention relates in part to the treatment and / or mitigation of coronavirus infection, including the treatment and / or mitigation of the inflammatory cytokine storm.

[0003] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, the entire contents of which are hereby incorporated by reference. The above ASCII copy was created on April 19, 2021, with the name "PNR - 001PC_ST25.txt" and a size of 3,951 bytes.

Background Art

[0004] Coronaviruses are a family of infectious, enveloped, positive-sense single-stranded RNA viruses, sometimes classified into four main types, including alpha, beta, gamma, and delta coronaviruses. (Fehr AR and Perlman S. Methods Mol Biol 2015;1282:1-23.) In December 2019, one such coronavirus disease was defined as coronavirus disease 19 (COVID-19), a novel severe acute respiratory syndrome caused by the SARS-CoV-2 virus. The SARS-CoV-2 virus (formerly known as 2019-nCoV) is closely related to SARS-CoV and has approximately 80% identical genomes. (Zhou P. et al. Nature 2020:10.1038.) COVID-19 and SARS-CoV share similar receptor-binding domain (RBD) structures and utilize the same cell entry receptor, ACE2. Li W. et al. Nature 2003;426:450-4.

[0005] The RBD domain of the COVID-19 S protein strongly interacts with the human ACE2 molecule, and it has been shown that ACE2 plays a crucial role in cell entry. Therefore, ACE2-expressing cells may act as target cells and are susceptible to COVID-19 infection. (Li W, et al. Nature 2003;426:450-4; Xu, X. et al. Science China Life Sciences vol 63,457-460 (2020)). High expression of ACE2 has been observed in type II alveolar cells (AT2) of the lung, upper esophageal cells and stratified epithelial cells, absorptive enterocytes of the ileum and colon, cholangiocarcinoma, cardiomyocytes, proximal renal tubular cells, and bladder urothelial cells. Xu,X.et al.Sci.China Life Sci.vol 63,457-460(2020);Zou,X.et al.Front Med.2020 Mar 12.doi:10.1007 / s11684-020-0754-0;Zhao,Y.et al.Clin Infect Dis.2020 Mar 28.pii:ciaa344.doi:10.1093 / cid / ciaa344.;Hao,Xu.et al.International J Oral Science(2020)12:8;Zhang,H.et al.Cell 181,April 16,2020;Chai,X.et al.2020 bioRxiv.doi:10.1101 / 2020.02.03.931766.

[0006] Many, though not all, viruses, including respiratory viruses such as influenza, respiratory rash viruses, rhinoviruses, and coronaviruses, suppress the innate immune response to gain an opportunity for efficient viral replication and establishment of infection. The result of the host's immune response is often incomplete, delayed, reduced, or, after a delay, very strong induction that can cause tissue damage. Kikkert M et al. J Innate Immun. 2020 Jan;12(1):4-20. It has been observed that seemingly mild cases of COVID-19 can rapidly worsen into severe cases affecting the lower lungs. The rapid deterioration of COVID-19 cases is thought to be partly due to a "cytokine storm," which is the overproduction of immune cells and their activating compounds (cytokines or chemokines), often accompanied by a surge of activated immune cells in the lungs and systemic inflammatory damage affecting many organs. In the lungs, the resulting pneumonia and edema (fluid accumulation) can lead to respiratory distress and may be exacerbated by secondary bacterial pneumonia, which increases the patient's risk of death. These patients may rapidly progress from acute respiratory distress syndrome (ARDS) and septic shock to multiple organ failure. Identifying and treating this excessive inflammation is crucial to reduce the increased mortality rate. Mehta et al. Lancet. 2020 Mar 28;395(10229):1033-1034. Most patients present with fever, dry cough, dyspnea, and bilateral ground-glass opacities on chest CT scans. Reports from COVID-19 patients have revealed that large amounts of IL-1β, IFNγ, IP10, and MCP1 may trigger an activated helper T1 (Th1) cell response. Severe COVID-19 patients also begin to secrete increased helper T2 (Th2) cytokines (e.g., IL-4 and IL-10), possibly as a countermeasure to suppress inflammation, and a strong association between cytokine storms and disease severity has been observed. Furthermore, patient test results identified increased levels of IL-6, in particular, as a risk factor for cytokine storms in patients with COVID-19 pneumonia.Cheung CY et al.2005.J Virol 79(12):7819-7826;Huang C,et al.Lancet 2020;395:497-506;Chen N,et al.Lancet 2020;395:507-13;Ruan et al.Intensive Care Med.2020 Mar 3.doi:10.1007 / s00134-020-05991-x.

[0007] These observations reveal a complex cytokine response during viral infection characterized by a series of overlapping networks. The cytokines TNF and IL-1β, as well as the chemotactic cytokines IL-8 and MCP-1, exhibit an acute response immediately following infection, followed by a sustained increase in IL-6. The interaction of IL-6 with its soluble receptors enhances IL-6 activity against target cells, further exacerbating inflammation. (Park WY, et al. 2001. Am. J. Respir. Crit. Care Med. 164:1896-1903.)

[0008] Compensatory repair processes are initiated as soon as an infection begins, attempting to restore tissue and organ function. Systemic production of IL-10 after a cytokine storm may serve as a marker of a suppressive anti-inflammatory response called "immunoparalysis," in that it is associated with the downregulation of neutrophil and mononuclear cell function in the systemic circulation. Downregulation of systemic inflammation may be conceptually beneficial in controlling the systemic response to a local infection. However, it has been suggested that patients who survived the initial cytokine storm but subsequently died were those who had not recovered from immunosuppression. Cohen J.2002.Nature 420:885-891;Fowler AA,et al.Am.J.Pathol.116:427-435;Tisonick JR.et al.Into the Eye of the Cytokine Storm.Microbiol Mol Biol Rev.2012 Mar;76(1):16-32;Munford RS,et al.2001.Am.J.Respir.Crit.Care Med.163:316-321.

[0009] Colony-stimulating factors (CSFs) refer to a family of four glycoproteins that control and regulate cell production through the deposition of widely dispersed bone marrow cells. These include granulocyte-macrophage CSF (GM-CSF), granulocyte colony CSF (G-CSF), macrophage colony CSF (M-CSF), and pluripotent colony-stimulating factor (IL-3). These lymphokines can induce progenitor cells found in the bone marrow to differentiate into specific types of mature blood cells. The specific type of mature blood cell that arises from the progenitor cells depends on the type of CSF present. See Metcalf D. Cancer Immunol Res. 2013, 1(6):351-356.

[0010] GM-CSF is a blood growth factor that regulates the production, migration, proliferation, differentiation, and function of hematopoietic cells. In response to inflammatory stimuli, GM-CSF is released by various cell types, including T lymphocytes, macrophages, fibroblasts, and endothelial cells. Subsequently, GM-CSF activates and enhances the production and survival of neutrophils, eosinophils, and macrophages. Native GM-CSF is typically produced near the site of action where it can regulate the proliferation, differentiation, and survival of hematopoietic progenitor cells. It is produced at picomolar concentrations (10⁻¹⁰ -10 ~10 -12 It is present in the circulating blood only in M). See Alexander WS. Int Rev Immunol. 1998, 16:651-682; Gasson JC. Blood. 1991, 77:1131-1145; Shannon MF et al. Crit Rev Immunol. 1997, 17:301-323; Barreda DR et al. Dev Comp Immunol. 2004, 28:509-554 and Metcalf D. Immunol Cell Biology. 1987, 65:35-43.

[0011] Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been approved by the FDA for use in combination with chemotherapy in the treatment of neutropenia, hematological disorders, and malignant neoplastic leukemia. Clinically, GM-CSF used to treat chemotherapy-induced neutropenia and aplastic anemia significantly reduces the risk of infection associated with bone marrow transplantation. Its usefulness in the treatment of myeloid leukemia and as a vaccine adjuvant is also well established. See Dorr RT. Clin Therapeutics. 1993. 15(1):19-29; Armitage JO. Blood 1998, 92:4491-4508; Kovacic JC et al. J Mol Cell Cardiol. 2007, 42:19-33; Jacobs PP et al. Microbial Cell Factories 2010, 9:93.

[0012] There are five classes of heterologous protein-producing platforms, including bacterial, yeast, plant, insect, and mammalian cells. However, more than 50% of currently marketed biopharmaceuticals are produced in mammalian cell lines. This is partly due to the fact that the remaining four classes cannot modify glycoproteins with human-like oligosaccharides. This is important because protein-binding glycans affect circulating half-life, tissue distribution, biological activity, and immunogenicity. LEUKINE is a yeast-derived recombinant humanized granulocyte-macrophage colony-stimulating factor (rhuGM-CSF, salglamostim) and is the only GM-CSF approved by the FDA.

[0013] Alveolar macrophages (AMs) are crucial for host defense against respiratory microorganisms. GM-CSF is an important cytokine that helps maintain healthy lungs. It contributes to the maturation of mononuclear phagocytes and AMs. GM-CSF deficiency (GM - / - ) Mouse-derived AM had impaired phagocytic and cytokine-producing capabilities, and these functions were restored by GM-CSF. - / -Mouse studies have shown that GM-CSF contributes to the immune response during pneumonia caused by Pseudomonas aeruginosa and Pneumocystis carinii, and that administration of GM-CSF to septic patients reverses mononuclear immunosuppression and improves their clinical course. Furthermore, GM-CSF has been shown to confer resistance to influenza by enhancing the innate immune mechanism dependent on alveolar macrophages. Human recombinant GM-CSF has also been shown to prevent fatal influenza infections in mice. Paine R 3rd et al.J Immunol.2000.164(5):2602-9;Paine R 3rd,et al.Am J Physiol Lung Cell Mol Physiol.2001.281(5):L1210-8;Shi Y et al.Cell Res.2006.16(2):126-33;Ballinger MN et al.Am J Respir Cell Mol Biol.2006.34(6):766-74;Meisel C et al.Am J Respir Crit Care Med 2009;180:640-648;Min L et al.J Immunol.2010.184(9):4625-9;Huang FF et al.Am J Respir Crit Care Med.2011.15;184(2):259-268.

[0014] Given the multipotency of GM-CSF, several studies suggest that inhibited GM-CSF may inhibit cytokine storm syndrome. (Spath S et al. Immunity 46,245-260; Sterner RM et al. Blood. 2019 Feb 14;133(7):697-709; Zhou Y et al. Perspective Immunol. 2020). I-Mab Biopharma has announced plans to develop TJM2 (TJ003234) for the treatment of cytokine storms caused by severe and critical COVID-19 infection. TJM2 is an antibody that neutralizes human granulocyte-macrophage colony-stimulating factor (GM-CSF). (https: / / www.pharmaceutical-technology.com / news / i-mab-covid-19-cytokine-storm-therapy / ). Interestingly, in a randomized phase II trial, GM-CSF treatment did not increase the number of ventilator-free days in patients with acute lung injury (ALI) or ARDS. Pain R 3 rd Crit Care Med. 2012 Jan;40(1):90-97. Therefore, at least some of the literature suggests that GM-CSF is ineffective or even harmful in situations of cytokine storm or lung injury.

[0015] Current management of COVID-19 is supportive, and respiratory failure in ARDS, often paired with cytokine storms, is a leading cause of death. There are no approved treatments for COVID-19, and while vaccines are available, their duration and applicability to variants are unknown. Therefore, there is an urgent need for treatments targeting SARS-CoV-2. Further treatments are needed that can prevent and / or reverse respiratory distress and / or cytokine storms associated with coronavirus infection. [Overview of the project]

[0016] Therefore, in one embodiment, the present invention relates to a method for treating coronavirus infection, comprising administering an effective amount of a composition containing granulocyte-macrophage colony-stimulating factor (GM-CSF) to a patient in need thereof.

[0017] In another embodiment, the present invention relates to a method for treating coronavirus infection, comprising administering an effective amount of a composition containing granulocyte-macrophage colony-stimulating factor (GM-CSF) to a patient in need, characterized in that the patient has a lower number of eosinophils compared to a non-infected state.

[0018] In yet another embodiment, the present invention provides a method for treating coronavirus infection, comprising: (a) selecting a patient who is infected with coronavirus and has one or more of the following conditions: (i) a low number of eosinophils compared to a non-infected state, (ii) a high level of ferritin compared to a non-infected state, and / or (iii) a high level of CRP compared to a non-infected state; and (b) administering to the patient an effective amount of a composition containing GM-CSF. [Brief explanation of the drawing]

[0019] [Figure 1] This shows the design of a randomized, open-label clinical trial. [Figure 2] This shows the absolute change in the alveolar-arterial (Aa) gradient in COVID-19 patients after treatment with LEUKINE. [Figure 3] This shows the absolute number of eosinophils in COVID-19 patients after treatment with LEUKINE. [Figure 4] This shows ferritin levels in COVID-19 patients after treatment with LEUKINE. [Figure 5] This shows the CRP levels of COVID-19 patients after treatment with LEUKINE. [Figure 6] This shows the lymphocyte count after treatment with LEUKINE. [Figure 7]Shows the levels of anti-SARS-CoV2 specific immunoglobulins after treatment with LEUKINE. Shows the effect of inhalation of LEUKINE on SARS-Cov2 specific immunoglobulins that are against spike (S1) and nucleocapsid (NCV) proteins. [Figure 8] Shows the number of HLA-DR+ CD38+ CD8+ T cells after treatment with LEUKINE. Shows the effect of inhalation of LEUKINE on the number of HLA-DR+ CD38+ CD8 T cells in PBMC. [Figure 9] Shows the number of activated CD8+ T cells after treatment with LEUKINE. Shows the effect of inhalation of LEUKINE on the number of IFNg+ and IL-2+ double positive CD8 T cells.

Mode for Carrying Out the Invention

[0020] The present invention relates, in part, to the surprising discovery that granulocyte macrophage colony-stimulating factor (GM-CSF) is an effective agent against coronavirus infections such as SARS and COVID-19, including treating and / or reversing cytokine storms associated with coronavirus infection and regulating the levels of immune cells.

[0021] Thus, in one aspect, the present invention provides a method for treating coronavirus infection.

[0022] Coronavirus Coronaviruses are members of the family Coronaviridae, which include beta- and alpha-coronavirus respiratory pathogens, and have only recently been known to infect humans. The family Coronaviridae includes beta-coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alpha-coronaviruses include, for example, HCoV-NL63 and HCoV-229E.

[0023] Coronaviruses enter host cells via a "spike" surface glycoprotein that facilitates viral entry into the host cell by angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor in mammalian hosts. (Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020). COVID-19, caused by SARS-CoV-2, is a novel virus thought to have originated in bats.

[0024] COVID-19 causes severe respiratory distress, and this RNA virus strain is responsible for the recent outbreak, which has been declared a major threat to public health and a global emergency. Phylogenetic analysis of the complete genome of SARS-CoV-2 has revealed that this virus is most closely related to the group of SARS-like coronaviruses (Betacoronavirus subgenus Salvecovirus) (89.1% nucleotide similarity). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020.

[0025] SARS-CoV-2 is an enveloped single-stranded RNA virus that encodes a "spike" protein, also known as the S protein, which is a surface glycoprotein; membrane-bound protein; envelope protein; and nucleocapsid protein that mediates binding to cell surface receptors. The S protein, including the S1 and S2 subunits, is a trimer class I fusion protein that exists in a pre-fusion conformation, undergoing structural rearrangement to fuse the viral membrane with the host cell membrane. For example, see Li, F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 3:237-261 (2016), the entire structure of which is incorporated herein by reference. The structure of the SARS-CoV-2 spike protein in the pre-fusion conformation has been discovered to date. See Daniel et al., Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation. Science, 19 Feb 2020, which is incorporated herein by reference in its entirety.

[0026] Phylogenetic analysis of the complete genome of SARS-CoV-2 (GenBank registration number: MN908947) revealed that this virus is most closely related to the SARS-like coronavirus group (Betacoronavirus genus, Salvecovirus subgenus) (89.1% nucleotide similarity). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020, which is incorporated herein by reference in its entirety.

[0027] SARS-CoV-2 possesses spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsidrin protein N. The complete genome (29,903 nucleotides, single-stranded RNA) of the SARS-CoV-2 coronavirus is listed in the NCBI database as GenBank reference sequence: MN908947. Coronavirus proteins can be selected from the group consisting of coronavirus spike protein (GenBank reference sequence: QHD43416), coronavirus membrane glycoprotein M (GenBank reference sequence: QHD43419), coronavirus envelope protein E (GenBank reference sequence: QHD43418), and coronavirus nucleocapsidrin protein E (GenBank reference sequence: QHD43423).

[0028] In some embodiments, the SARS-CoV-2 coronavirus is the "Wuhan strain" or a variant strain. In some embodiments, the variant strain is one or more of B.1.1.7, B1.351, B.1, B.1.1.28, B.1.2, CAL.20C, B.6, P.1, and P.2, as well as / or any other variant or its antigenic fragment. In some embodiments, the variant strain is one or more of A.1, A.2, A.3, A.4, A.5, A.6, A.7, A.8, A.9, B, B.1, B.1.1, B.1.1.1, B.2, B.3, B.4, B.5, B.6, B.7, B.9, B.10, B.11, B.12, B.13, B.14, B.15, B.16, B.17, B.18, B.19, B.20, B.21, B.22, B.23, B.24, B.25, B.26, B.27, C.1, C.2, C.3, D.1, and D2. In some embodiments, the variant strain has one or more mutations compared to the Wuhan strain, for example, D614G, E484K, N501Y, K417N, S477G, and S477N.

[0029] Composition of GM-CSF The GM-CSF used in carrying out the present invention includes any pharmaceutically safe and effective GM-CSF, or any derivative thereof having the biological activity of GM-CSF. In one embodiment, the GM-CSF used in carrying out the method of the subject is recombinant human GM-CSF (rhu GM-CSF), such as salglamostim (LEUKINE). Salglamostim is a recombinant human GM-CSF derived from biosynthetic yeast, having a single 127-amino acid glycoprotein, which differs from endogenous human GM-CSF in that it has leucine instead of proline at position 23. Other natural and synthetic GM-CSFs and derivatives thereof having the biological activity of natural human GM-CSF may be equally useful in carrying out the present invention.

[0030] In embodiments, GM-CSF is produced or can be produced in bacterial, yeast, plant, insect, and mammalian cells. In embodiments, GM-CSF is produced or can be produced in Escherichia coli cells. In embodiments, GM-CSF is produced or can be produced in yeast cells. In embodiments, GM-CSF is produced or can be produced in Chinese hamster ovary cells (CHO). In embodiments, GM-CSF is not produced in E. coli cells. In embodiments, GM-CSF is produced in cells that enable glycosylation, such as yeast or CHO cells.

[0031] In an embodiment, GM-CSF has the amino acid sequence of SEQ ID NO: 1, or a variant that is approximately 90%, 93%, 95%, 97%, or 98% identical thereto. In an embodiment, GM-CSF has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a variant that is approximately 90%, 93%, 95%, 97%, or 98% identical thereto. In an embodiment, GM-CSF is one of morglamostim, salglamostim, and regramostim.

[0032] While we do not wish to be constrained by theory, the core of hGM-CSF consists of four helices arranged at angles. Crystal structure and mutagenicity analysis of rhGM-CSF (Rozwarski DA et al., Proteins 26:304-13, 1996) revealed that, in addition to the nonpolar side chains of the protein core, 10 embedded hydrogen-bonding residues are involved in intramolecular hydrogen bonding to the main chain atoms, which is better conserved than residues that hydrogen-bond to other side chain atoms; 24 solvation sites were observed at equivalent positions of two molecules in the asymmetric unit, with the strongest of these located in the gaps between secondary structure elements. Two surface clusters of hydrophobic side chains are located near the expected receptor-binding region. Mutagenesis of residues on the helix A / helix C surface confirmed the importance of specific Glu, Gly, and Gln residues. Therefore, these residues are not substituted in the functional substitution variants of hGM-CSF used in the present invention, and these helices are maintained in the functional fragments or deletion variants of hGM-CSF used in the present invention. Furthermore, in embodiments, those skilled in the art can refer to UniProtKB entry P04141 for structural information to report the identity of the variant.

[0033] The N-terminal helix of hGM-CSF governs its high-affinity binding to its receptor (Shanafelt AB et al., EMBO J 10:4105-12, 1991). The transformation of GM-CSF's biological effects requires interaction with at least two cell surface receptor components (one of which is shared with the cytokine IL-5). In the above study, receptor-binding determinants within GM-CSF were identified by locating unique receptor-binding domains on a series of human-mouse hybrid GM-CSF cytokines. The interaction between GM-CSF and the shared subunits of their high-affinity receptor complexes was governed by a very small portion of the peptide chain. The presence of several key residues in the N-terminal α-helix was sufficient to confer specificity to the interaction.

[0034] In some embodiments, amino acid mutations are amino acid substitutions, which may include conserved substitutions and / or non-conservative substitutions.

[0035] "Conservative substitutions" may be made, for example, based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0036] As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid enumerated within the same group of the six standard amino acid groups described above. For example, the exchange of Asp by Glu retains one negative charge within the thus modified polypeptide. Furthermore, glycine and proline may be substituted for each other based on their ability to disrupt α-helices.

[0037] As used herein, “non-conservative substitution” is defined as the exchange of an amino acid by another amino acid listed in a different group from the six standard amino acid groups (1) to (6) above.

[0038] In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolicin, N-formylmethionine β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, salcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids, e.g., β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and common amino acid analogs).

[0039] Amino acid sequence modifications can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0040] While we do not wish to be constrained by theory, the degree of glycosylation of biosynthesized GM-CSF appears to affect its half-life, distribution, and exclusion. (Lieschke and Burgess, N.Engl.J.Med.327:28-35,1992; Dorr, RT, Clin.Ther.15:19-29,1993; Horgaard et al., Eur.J.Hematol.50:32-36,1993). In embodiments, the GM-CSF molecule of the present invention is glycosylated.

[0041] Treatment methods In one embodiment, the present invention relates to a method for treating coronavirus infection, comprising administering an effective amount of a composition containing granulocyte-macrophage colony-stimulating factor (GM-CSF) to a patient in need.

[0042] In another embodiment, the present invention relates to a method for treating coronavirus infection, comprising administering an effective amount of a composition containing granulocyte-macrophage colony-stimulating factor (GM-CSF) to a patient in need, characterized in that the patient has a lower number of eosinophils compared to a non-infected state.

[0043] In another embodiment, the present invention provides a method for treating coronavirus infection, comprising: (a) selecting a patient who is infected with coronavirus and has one or more of the following conditions: (i) a low number of eosinophils compared to a non-infected state, (ii) a high level of ferritin compared to a non-infected state, and / or (iii) a high level of CRP compared to a non-infected state; and (b) administering to the patient an effective amount of a composition containing GM-CSF.

[0044] In embodiments, the method further includes the step of monitoring the number of eosinophils during treatment. In embodiments, an increase in the number of eosinophils leads to continued administration of GM-CSF. In embodiments, a decrease in the number of eosinophils leads to discontinuation of GM-CSF administration.

[0045] In embodiments, the method further includes the step of monitoring ferritin levels during treatment. In embodiments, a decrease in ferritin levels leads to continued administration of GM-CSF. In embodiments, an increase in ferritin levels leads to discontinuation of GM-CSF administration.

[0046] In embodiments, the method further includes the step of monitoring CRP levels during treatment. In embodiments, a decrease in CRP levels leads to continued administration of GM-CSF. In embodiments, an increase in CRP levels leads to discontinuation of GM-CSF administration.

[0047] In the embodiment, the number of eosinophils, ferritin levels, and / or CRP levels are measured in the patient's biological sample.

[0048] In embodiments, the coronavirus is selected from (i) betacoronaviruses optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1 and HCoV-OC43, and (ii) alphacoronaviruses optionally selected from HCoV-NL63 and HCoV-229E. In embodiments, the coronavirus is SARS-CoV-2.

[0049] In an embodiment, the patient has COVID-19. In an embodiment, the patient has one or more of the following symptoms: fever, cough, shortness of breath, diarrhea, upper respiratory tract disorder, lower respiratory tract disorder, pneumonia, and acute respiratory distress. In an embodiment, the patient is hypoxic. In an embodiment, the patient is suffering from respiratory distress.

[0050] In embodiments, the method increases the number of eosinophils in a patient. In embodiments, the number of eosinophils is measured in a biological sample from the patient. In embodiments, the biological sample includes blood, respiratory fluid, saliva, or feces. In embodiments, respiratory fluid is from an oropharyngeal (OP) or nasopharyngeal (NP) swab. In embodiments, respiratory fluid is a lavage solution, which may include a bronchial lavage solution. In embodiments, respiratory fluid is sputum. In embodiments, respiratory fluid is nasal secretions. In embodiments, respiratory fluid is saliva.

[0051] In embodiments, the method of the present invention is used in patients receiving treatment with one or more steroids, such as corticosteroids, such as methylprednisone, and optionally steroids administered orally or by injection. While not wishing to be bound by theory, steroid-induced eosinophilia can be corrected by GM-CSF administration according to the present invention. Therefore, in embodiments, patients can continue steroid treatment in the context of the method of the present invention.

[0052] In some embodiments, this method prevents or alleviates the onset of acute respiratory distress syndrome (ARDS) in patients. In some embodiments, this method improves oxygenation in patients.

[0053] In this embodiment, the method reduces the viral load in the patient compared to before treatment.

[0054] In embodiments, the method prevents or mitigates the transition from respiratory distress to cytokine imbalance in a patient. In embodiments, the method reverses or prevents a cytokine storm. In embodiments, the method reverses or prevents a cytokine storm in the lung or systemically. In embodiments, the cytokine storm is selected from one or more of the following: systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistitis. In embodiments, the method reverses or prevents the overproduction of one or more inflammatory cytokines. In embodiments, the inflammatory cytokine is one or more of the following: IL-6, IL-1, IL-2, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

[0055] In embodiments, the method reduces ferritin levels compared to pre-treatment levels. In embodiments, the method reduces ferritin to less than approximately 1000 ng / ml, and if necessary, to less than approximately 650 ng / ml. In embodiments, the method reduces C-reactive protein (CRP) compared to pre-treatment levels. In embodiments, the method reduces CRP to less than approximately 10 mg / L, and if necessary, to less than approximately 3 mg / L.

[0056] In embodiments, the method improves the quantity and / or quality of SARS-CoV-2 antigen-specific T cells, such as cytotoxic T cells or helper T cells, in patients (for example, when measured by anti-infective effect) compared to an untreated or pre-treatment state.

[0057] In embodiments, the method improves the quantity and / or quality of SARS-CoV-2 antigen-specific CD8+ T cells in a patient (for example, when measured by anti-infective effect) (compared to, for example, an untreated or pre-treatment state).

[0058] In embodiments, the method improves the quantity and / or quality of SARS-CoV-2 antigen-specific activated CD8+ T cells in a patient (for example, when measured by anti-infective effect) (compared to, for example, an untreated or pre-treatment state).

[0059] In embodiments, the method improves the quantity and / or quality of SARS-CoV-2 antigen-specific HLD-DR+ CD38+ CD8+ T cells (for example, when measured by anti-infective effect) compared to an untreated or pre-treatment state.

[0060] In embodiments, the method improves the quantity and / or quality of SARS-CoV-2 antigen-specific IFN-γ and IL-2 secreting CD8+ T cells (for example, when measured by anti-infective effect) compared to an untreated or pre-treatment state.

[0061] In embodiments, the method modulates migratory dendritic cells (DCs). In embodiments, these migratory DCs activate SARS-CoV-2 antigen-specific CD8+ T cells in a patient, such as HLD-DR+ CD38+ CD8+ T cells and / or IFN-γ and IL-2 secreting CD8+ T cells (compared to, for example, an untreated or pre-treatment state).

[0062] In embodiments, the method modulates alveolar macrophages (AMs). In embodiments, these AMs activate SARS-CoV-2 antigen-specific CD8+ T cells in the patient, e.g., HLD-DR+ CD38+ CD8+ T cells and / or IFN-γ and IL-2 secreting CD8+ T cells (compared to, for example, an untreated or pre-treatment state).

[0063] In one embodiment, the present invention provides a method for inducing a SARS-CoV-2 antigen-specific immune response in a subject by administering the GM-CSF agent of the present invention. In this embodiment, the SARS-CoV-2 antigen-specific immune response is mediated by one or more CD8+ T cells, AMs, and DCs.

[0064] In embodiments, the GM-CSF of the present invention enhances the innate immune response, i.e., modulates AM, thereby providing the therapeutic agent of the present invention for coronavirus infection.

[0065] In some embodiments, the methods of the present invention provide a treatment for patients who have not been vaccinated or are unable to be vaccinated. In some embodiments, the methods of the present invention provide a treatment for patients who have been vaccinated but are still infected (for example, due to ineffective vaccination and / or a decrease in the effectiveness of the vaccine over time). In some embodiments, the coronavirus vaccine comprises one or more of the following: attenuated live virus, inactivated virus, non-replicating viral vector, replicating viral vector, recombinant protein, peptide, virus-like particle, DNA, RNA, mRNA, another macromolecule, and fragments thereof. In some embodiments, the coronavirus vaccine is selected from mRNA-1273, AZD1222, BNT162, Ad5-nCoV, INO-4800, and LV-SMENP-DC, as well as pathogen-specific aAPC, or variants or derivatives thereof. In some embodiments, the coronavirus vaccine comprises an LNP-encapsulated mRNA vaccine, such as mRNA-1273, which encodes the SARS-CoV-2 spike (S) protein, and optionally includes the like. In some embodiments, the coronavirus vaccine includes a viral vector vaccine expressing the S protein, a viral vector (ChAdOx1-chimpanzee adenovirus Oxford 1) vaccine expressing the S protein as needed (ChAdOx1 nCoV-19), AZD1222, etc. In some embodiments, the coronavirus vaccine includes an mRNA vaccine encoding an optimized SARS-CoV-2 RBD, BNT162b1, etc. In some embodiments, the coronavirus vaccine includes an mRNA vaccine encoding an optimized full-length S protein, BNT162b2, etc. In some embodiments, the coronavirus vaccine includes an adenovirus type 5 vector expressing a protein selected from spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsidrin protein N, an adenovirus type 5 vector expressing the S protein as needed, Ad5-nCoV, etc.In some embodiments, the coronavirus vaccine includes a plasmid encoding an S protein introduced by electroporation, a DNA plasmid encoding an S protein introduced by electroporation if necessary, such as INO-4800. In some embodiments, the coronavirus vaccine includes dendritic cells (DCs) modified with a lentiviral vector expressing a synthetic minigene based on a selected viral protein domain, such as LV-SMENP-DC, administered together with antigen-specific cytotoxic T lymphocytes (CTLs). In some embodiments, the coronavirus vaccine includes artificial antigen-presenting cells (aAPCs) modified with a lentiviral vector expressing a synthetic minigene based on a selected viral protein domain, such as pathogen-specific aAPCs.

[0066] Pharmacopoeia-acceptable salts and excipients The compositions described herein may have sufficient basic functional groups that can react with inorganic or organic acids, or carboxyl groups that can react with inorganic or organic bases, in order to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as is well known in the art. Examples of such salts include those listed in the Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PHStahl and CGWermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0067] Pharmaceutically acceptable salts, in non-limiting examples, include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bicarbonate tartrates, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, glucaronic acid, sugars, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, pamoate, phenylacetates, trifluoroacetates, acrylates, chlorobenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, methylbenzoates, and o-acetoxybenzoates. Examples include benzoates, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butin-1,4-dicarboxylate, hexin-1,4-dicarboxylate, caprine, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenyl propionate, sebacinate, suberinate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate salts.

[0068] The term "pharmaceutically acceptable salt" also refers to salts of the compositions of the present invention having acidic functional groups such as carboxylic acid functional groups and bases. Suitable bases include, but are not limited to, alkali metal hydroxides such as sodium, potassium, and lithium; alkaline earth metal hydroxides such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxysubstituted mono, di, or trialkylamines and dicyclohexylamines; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono, bis, or tris(2-OH lower alkylamines) such as mono-, bis-, or tris-(2-hydroxyethyl)amine; 2-hydroxy-tert-butylamine; or tris-(hydroxymethyl)methylamine; N,N-dimethyl-N-(2-hydroxyethyl)amine; or tris-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine.

[0069] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0070] Pharmaceutical compositions and preparations In various embodiments, the present invention relates to pharmaceutical compositions comprising a composition, for example, GM-CSF and / or additional therapeutic agents as described herein, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions described herein may be administered to a subject as components of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally contain appropriate amounts of pharmaceutically acceptable excipients to provide a form for appropriate administration.

[0071] In various embodiments, the pharmaceutically acceptable excipients may be liquids such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Examples of pharmaceutically acceptable excipients include saline solution, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, and urea. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to the subject. Water is a useful excipient when any of the drugs described herein are administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid excipients, particularly for infusion solutions. Other suitable pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. Any of the agents described herein may also contain small amounts of wetting or emulsifying agents or pH buffers, as needed. Other examples of suitable pharmaceutically acceptable excipients are given in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0072] The present invention comprises the pharmaceutical compositions (and / or additional therapeutic agents) described herein in various formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may take the form of a solution, suspension, emulsion, infusion, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, freeze suspension, dehydrated powder, or other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a softgel capsule. In yet another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0073] If necessary, the pharmaceutical composition (and / or additional therapeutic agent) of the present invention may also contain a solubilizer. The agent can also be delivered using a suitable vehicle or delivery device known in the art. The combination therapeutic agents outlined herein can be delivered in combination using a single delivery vehicle or delivery device.

[0074] Formulations comprising the pharmaceutical composition (and / or additional therapeutic agents) of the present invention may preferably be provided in unit dosage forms and may be prepared by any method well known in the pharmaceutical field. Such methods generally involve the step of associating a therapeutic agent with a carrier constituting one or more minor components. Typically, formulations are prepared by homogeneously and closely associating a therapeutic agent with a liquid carrier, a pulverized solid carrier, or both, and the product is then, if necessary, molded into the dosage form of the desired formulation (e.g., wet or dry granulation, powder blending, etc., and then tableted using conventional methods known in the art).

[0075] In various embodiments, any pharmaceutical composition (and / or additional therapeutic agent) described herein is formulated according to conventional procedures as a composition suitable for the administration method described herein.

[0076] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, vaginal, transdermal, rectal, inhalation, or local routes. Administration may be local or systemic. In some embodiments, administration is performed orally. In other embodiments, administration is performed by parenteral injection. The method of administration is left to the discretion of the healthcare professional and may depend in part on the site of the medical condition. In most cases, administration results in the release of any of the drugs described herein into the bloodstream.

[0077] In certain embodiments, GM-CSF (and / or additional therapeutic agents) is administered via an intravenous route.

[0078] In certain embodiments, GM-CSF (and / or additional therapeutic agents) is administered into the lungs.

[0079] In certain embodiments, GM-CSF (and / or additional therapeutic agents) is administered via aerosol or nebulizer.

[0080] In certain embodiments, the aerosol or nebulizer is selected from liquid spray, dry powder dispersion, and dose-measured administration. In certain embodiments, the aerosol or nebulizer is selected from jet stream or mesh vibration.

[0081] In certain embodiments, GM-CSF (and / or additional therapeutic agents) is administered by inhalation. While we do not wish to be bound by theory, inhalation of salglamostim reduces the patient's systemic exposure.

[0082] In embodiments, GM-CSF (and / or additional therapeutic agents) are administered by inhalation and mediate local and / or peripheral cellular responses. For example, in embodiments, GM-CSF (and / or additional therapeutic agents) are administered by inhalation and mediate an increase in lymphocytes and / or eosinophils in the peripheral blood.

[0083] Therefore, in embodiments, a method is provided for modulating peripheral immune cells to induce an anti-infective effect via local administration (e.g., inhalation).

[0084] In one embodiment, the pharmaceutical compositions (and / or additional therapeutic agents) described herein are formulated as compositions suitable for oral administration by conventional procedures. Orally administered compositions may be, for example, in the form of tablets, oral tablets, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as sweeteners (e.g., fructose, aspartame, or saccharin); flavoring additives (e.g., peppermint, madder oil, or cherry); colorants; and preservatives to obtain a pharmaceutically pleasing preparation. Furthermore, in the form of tablets or pills, the compositions may be coated to slow disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. A selectively permeable membrane surrounding any of the pharmaceutical compositions (and / or additional therapeutic agents) described herein, driven by permeable activity, is also suitable for orally administered compositions. In these latter platforms, the fluid in the environment surrounding the capsule is absorbed by the driving compound, which then expands and replaces the drug or drug composition through the opening. These delivery platforms can provide an essentially zero-order delivery profile, in contrast to the spike profile of immediate-release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate may also be useful. The oral composition may contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are pharmaceutical grade. In addition to the active compound, the suspension may contain anti-precipitation agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxyoxide, bentonite, agar, tragacanth, and mixtures thereof.

[0085] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, and emulsions. They can also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injection medium immediately before use. They may contain, for example, suspensions or dispersants known in the art. Suitable formulation components for parenteral administration include sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetates, citrates, or phosphates; and isotonic modifiers such as sodium chloride or dextrose.

[0086] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under manufacturing and storage conditions and must be protected from microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

[0087] The compositions described herein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., "sprays") administered by inhalation. The aerosol formulations can be contained in pressurized sprays such as dichlorodifluoromethane, propane, or nitrogen.

[0088] The pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may be administered by controlled-release or sustained-release means or by delivery devices well known to those skilled in the art. Examples include, but are not limited to, those described in U.S. Patents No. 3,845,770, No. 3,916,899, No. 3,536,809, No. 3,598,123, No. 4,008,719, No. 5,674,533, No. 5,059,595, No. 5,591,767, No. 5,120,548, No. 5,073,543, No. 5,639,476, No. 5,354,556, and No. 5,733,556, each of which is incorporated herein by reference in whole. Such dosage forms may be useful for providing controlled or sustained release of one or more active ingredients by using, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microparticles, or combinations thereof, to provide desired release profiles in various proportions. Suitable controlled-release or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the drugs described herein. Accordingly, the present invention provides single unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps, and caplets, adapted for controlled or sustained release.

[0089] The controlled or sustained release of the active ingredient may be stimulated by a variety of conditions, including but not limited to changes in pH, temperature, stimulation by light of an appropriate wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0090] In another embodiment, the controlled-release device can be positioned near the target area being treated, and therefore requires only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, ibid., vol. 2, pp. 115-138 (1984)). Other controlled-release devices may be used, as discussed in the review by Langer, 1990, Science 249:1527-1533.

[0091] Pharmaceutical preparations are preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. If the composition is freeze-dried, filter sterilization can be performed before or after freeze-drying and solution preparation.

[0092] Dosage and Administration It will be understood that the actual dose of the composition administered according to the present invention will vary depending on the specific dosage form and method of administration. Many factors that may alter the action of the composition (e.g., body weight, sex, diet, timing of administration, route of administration, excretion rate, subject condition, drug combination, genetic predisposition, and response sensitivity) can be taken into consideration by those skilled in the art. Administration can be carried out continuously, or in single or multiple individual doses, within the range of the maximum tolerated dose. The optimal administration rate for a given set of conditions can be determined by those skilled in the art using conventional dose-dosage tests.

[0093] In embodiments, GM-CSF is administered in a total dose of approximately 125 μg, 150 μg, 200 μg, 250 μg, 300 μg, or 350 μg. In embodiments, GM-CSF is administered in a total dose of approximately 250 μg.

[0094] In embodiments, GM-CSF is administered in doses of approximately 125 μg, 150 μg, 200 μg, 250 μg, 300 μg, or 350 μg.

[0095] In this embodiment, GM-CSF is administered twice daily.

[0096] In this embodiment, GM-CSF is salglamostim administered twice daily at a dose of approximately 125 μg.

[0097] Combination therapy and additional therapeutic agents In various embodiments, the pharmaceutical compositions of the present invention are administered in combination with additional drugs(s). This combination administration can be carried out simultaneously or sequentially.

[0098] In one embodiment, the additional therapeutic agent and the GM-CSF of the present invention are administered to the subject simultaneously. The term “simultaneously,” as used herein, means that the additional therapeutic agent and the GM-CSF are administered at time intervals of no more than 60 minutes, such as within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute. The administration of the additional therapeutic agent and the GM-CSF may be by simultaneous administration of a single formulation (e.g., a formulation containing the additional therapeutic agent and the GM-CSF composition) or separate formulations (e.g., a first formulation containing the additional therapeutic agent and a second formulation containing the GM-CSF composition).

[0099] Concomitant administration does not require simultaneous administration of the therapeutic agents if the timing of administration allows for a temporal overlap in the pharmacological activity of the additional therapeutic agent and GM-CSF, thereby producing a combined therapeutic effect. For example, the additional therapeutic agent and the GM-CSF composition, which is a targeted component, can be administered sequentially. As used herein, the term “sequentially” means that the additional therapeutic agent and GM-CSF are administered at time intervals of more than approximately 60 minutes. For example, the time between sequential administrations of the additional therapeutic agent and GM-CSF may be more than approximately 60 minutes, more than approximately 2 hours, more than approximately 5 hours, more than approximately 10 hours, more than approximately 1 day, more than approximately 2 days, more than approximately 3 days, more than approximately 1 week, more than approximately 2 weeks, or more than approximately 1 month. The optimal administration time depends on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and GM-CSF being administered. Either the additional therapeutic agent or the GM-CSF composition may be administered first.

[0100] Concomitant administration does not require that the therapeutic agents be administered to the subject via the same route of administration. Rather, each therapeutic agent can be administered via an appropriate route, for example, orally or parenterally.

[0101] In some embodiments, the GM-CSF described herein acts synergistically when administered in combination with another therapeutic agent. In such embodiments, the GM-CSF composition, which is a targeted component, and the additional therapeutic agent can be administered in lower doses than those used when the agent is used in conjunction with monotherapy.

[0102] In some embodiments, additional therapeutic agents are selected from remdesivir; favipiravir; galidesivir; prezcobix; lopinavir; and / or ritonavir; and / or arbidol-lopinavir / ritonavir; and / or ribavirin; and / or IFN-β; xiyanping; anti-VEGF-A; fingolimod; kalimycin; hydroxychloroquine; darunavir and cobicistat; methylprednisolone; brilacidine; leronlimab; and thalidomide.

[0103] In some embodiments, the additional therapeutic agent is an antibody, such as a monoclonal antibody, that counteracts the coronavirus antigen, such as the spike protein, such as its RBD. In some embodiments, the additional therapeutic agent is one or more of bamranivimab, casirivimab, and imdevimab.

[0104] Methods for detecting therapeutic effects In some embodiments, methods are provided for detecting the success or potential response to treatment with the GM-CSF agent of the present invention for coronavirus infection. In embodiments, methods are provided for detecting the success or potential response to treatment with the GM-CSF agent of the present invention for coronavirus infection by using KL-6 as a biomarker. In embodiments, the GM-CSF agent of the present invention reduces KL-6 in subjects suffering from coronavirus infection, and as a result, this reduction is used as a biomarker for treatment. For example, in embodiments, KL-6 levels are determined (for example, when measured via protein or nucleic acid levels in a biological sample, e.g., blood, respiratory fluid (e.g., from oropharyngeal (OP) or nasopharyngeal (NP) swabs or lavage fluids, where the lavage fluids may include bronchial lavage fluids and further include sputum, nasal secretions or saliva)), saliva, or feces). In some embodiments, a decrease in KL-6 (e.g., compared to before treatment or untreated) signifies treatment success, while a small decrease in KL-6 or an increase in KL-6 signifies less treatment success (e.g., leading to higher doses and / or alternative treatments (e.g., one or more of those disclosed herein)).

[0105] In some embodiments, methods are provided for detecting the likelihood of successful treatment with the GM-CSF agents of the present invention against coronavirus infection by measuring the presence, absence, or level of one or more MUC1 agents, including KL-6, etc. For example, in embodiments, methods are provided for measuring the presence, absence, or level of one or more MUC1 agents, including KL-6, etc., by taking a sample from a patient who has or is suspected of having a coronavirus infection, etc., including KL-6, etc., where (i) high levels (e.g., compared to normal or untreated levels) indicate a high likelihood of successful treatment and lead to treatment or continued treatment with the GM-CSF agents of the present invention, or (ii) low levels (e.g., compared to normal or untreated levels) indicate a low likelihood of successful treatment and lead to treatment with an alternative therapy to the GM-CSF agents of the present invention.

[0106] In some embodiments, methods are provided for detecting a response to treatment with the GM-CSF agents of the present invention against coronavirus infection by measuring the presence, absence, or level of one or more MUC1 agents, including KL-6. For example, in embodiments, methods are provided for measuring the presence, absence, or level of one or more MUC1 agents, including KL-6, by taking a sample from a patient who has or is suspected of having a coronavirus infection and is being treated with the GM-CSF agents of the present invention, and measuring the presence, absence, or level of one or more MUC1 agents, including KL-6, including KL-6, where (i) a low level (e.g., compared to a normal level or untreated level) means a positive response to treatment leading to continued treatment with the GM-CSF agents of the present invention, or (ii) a high level (e.g., compared to a normal level or untreated level) means a negative response to treatment leading to treatment with an alternative agent.

[0107] Use of Morglamostim In some embodiments, the present invention relates to the use of morglamostim in methods of treating or preventing infection by administration via inhalation and, if necessary, by modulating the peripheral immune response (e.g., by modulating antigen-specific CD8+ T cells in a patient, e.g., HLD-DR+ CD38+ CD8+ T cells, and / or IFN-γ and IL-2 secreting CD8+ T cells (e.g., compared to an untreated or pre-treatment state)).

[0108] In embodiments, such morglamostim is used in methods for treating or preventing viral, parasitic, or bacterial infections.

[0109] In various embodiments, the present invention provides a method for treating a viral infection using morphulamostim, wherein the coronavirus causing the viral infection is selected from (i) betacoronaviruses optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1 and HCoV-OC43, and (ii) alphacoronaviruses optionally selected from HCoV-NL63 and HCoV-229E. In embodiments, the coronavirus is SARS-CoV-2.

[0110] In various embodiments, the present invention provides a method for treating viral infections using Morglamostim, including but not limited to acute or chronic viral infections of internal organs, such as respiratory tract, papillomavirus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis virus infections. In some embodiments, the viral infection is caused by a virus of the family Flaviviridae. In some embodiments, the virus of the family Flaviviridae is selected from yellow fever virus, West Nile virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, and hepatitis C virus. In other embodiments, the viral infection is caused by a virus of the family Picornaviridae, such as poliovirus, rhinovirus, and coxsackievirus. In other embodiments, the viral infection is caused by a member of the family Orthomyxoviridae, such as influenza virus. In other embodiments, the viral infection is caused by a member of the family Retroviridae, such as lentivirus. In other embodiments, the viral infection is caused by members of the Paramyxoviridae family, such as respiratory rash virus, human parainfluenza virus, rubra virus (e.g., mumps virus), measles virus, and human metapneumovirus. In other embodiments, the viral infection is caused by members of the Bunyaviridae family, such as hantavirus. In other embodiments, the viral infection is caused by members of the Reoviridae family, such as rotavirus.

[0111] In various embodiments, the present invention provides a method for treating parasitic infections, such as protozoan or helminthic infections, using morglamostim. In some embodiments, the parasitic infection is caused by a protozoan parasite. In some embodiments, the oritiziab parasite is selected from enteroprotozoa, histoprotozoa, or hematoprotozoa. Examples of protozoan parasites include, but are not limited to, Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. In some embodiments, the parasitic infection is caused by helminthic parasites such as nematodes (e.g., Adenophorea). In some embodiments, the parasite is selected from the biglanta class (e.g., Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis). In some embodiments, the parasite is selected from the trematodes class (e.g., Schistosoma, Hepatophora, Enterophora, and Lung flukes).In some embodiments, the parasite is selected from Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes, and Paragonimus westermani. In some embodiments, the parasite is selected from tapeworms (e.g., Taenia solium, Taenia saginata, Hymenolepis nana, Echinococcus granulosus).

[0112] In various embodiments, the present invention provides a method for treating bacterial infections using Morglamostim. In various embodiments, the bacterial infection is caused by Gram-positive bacteria, Gram-negative bacteria, aerobic bacteria, and / or anaerobic bacteria. In various embodiments, the bacteria are selected from, but are not limited to, Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter, Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus, Brucella, and other microorganisms. In some embodiments, the bacteria include, but are not limited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, ProvidenciaAlcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridiumdifficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, or Staphylococcus saccharolyticus.

[0113] array Sequence ID 1 is wild-type GM-CSF: APARSPSPSTQPWEHVNAIQEAPRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE is Sequence ID 2 is salglamostim: APARSPSPSTQPWEHVNAIQEALRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE is Sequence ID 3 is Morglamostim: APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE is

[0114] definition The following definitions are used in connection with the inventions disclosed herein. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the inventions belong.

[0115] "Effective dose" is the amount that is effective in treating or alleviating a coronavirus infection, when used in relation to a drug that is effective in treating a coronavirus infection.

[0116] As used herein, "a" or "the" may mean one or more. Furthermore, when used in relation to a referenced numerical representation, the term "about" means adding or subtracting up to 10% of that referenced numerical representation. For example, the phrase "about 50" encompasses the range from 45 to 55.

[0117] As used herein, all proportions of compositions are relative to the total weight of the composition unless otherwise specified. Where used herein, the word “include” and its variations are intended to be non-limiting, and the descriptions of items in the list do not exclude other similar items that may be useful in the materials, compositions, devices, and methods of this art. Similarly, the terms “can” and “may” and their variations are intended to be non-limiting, and any description that an embodiment may include or may include certain elements or features does not exclude other embodiments of the art that do not include those elements or features.

[0118] The non-restrictive term "comprising," which is synonymous with the terms "including," "containing," or "having," is used herein to describe and claim an invention, the present invention, or its embodiments, but may be described alternatively using alternative terms such as "consisting of" or "consisting essentially of."

[0119] The present invention is further illustrated by the following non-limiting embodiments. [Examples]

[0120] Example 1: Clinical trial design of salglamostim (LEUKINE) for the treatment of COVID-19 A prospective, randomized, open-label intervention study was conducted to investigate the effects of salglamostim (LEUKINE) on oxygenation and improvement of short-term and long-term outcomes in COVID-19 patients with acute hypoxic respiratory failure (Figure 1).

[0121] For prospective participants aged 18–80 years, we identified whether they met the clinical, radiological, and research criteria for ARDS if they exhibited symptoms of COVID-19 infection. Patients were divided into two groups: Group A, who received standard treatment plus 125 μcg of salglamostim inhaled twice daily (IH); and Group B, who received standard treatment only on days 1–5 (D1–5). After D5, if a patient progressed to ARDS with mechanical ventilation, such patients in both groups (Group C and Group D, respectively) were administered 125 μcg / m². 2 Salglamostim was administered intravenously (IV). The patient will be monitored for a further 10-20 weeks after treatment.

[0122] Exclusion criteria included patients with known hypersensitivity to human granulocyte-macrophage colony-stimulating factors, such as salglamostim (GM-CSF), yeast-derived products, or any component of LEUKINE; patients enrolled in other clinical trials; pregnant or lactating patients; patients with peripheral blood white blood cell counts exceeding 25,000 per ml and / or active malignancies; patients receiving high doses of systemic steroids (more than 20 mg of methylprednisolone or equivalent) or lithium carbonate therapy; and patients with serum ferritin levels exceeding 2000 μcg / ml.

[0123] The primary objective was to investigate whether inhaled LEUKINE administration for the first five days improved oxygenation in COVID-19 patients. A secondary objective was to determine whether early intervention with LEUKINE had a favorable safety profile and affected progression to mechanical ventilation and / or ARDS.

[0124] Example 2: Clinical Results Administration of LEUKINE to COVID-19 infected patients reduced the alveolar-arterial (Aa) absolute gradient from D1 to D6 compared to the state of care (SOC). This suggests that LEUKINE administration to patients initiated the normalization of some abnormality in oxygen diffusion into the blood caused by COVID-19 (Figure 2). Furthermore, treatment with LEUKINE increased the absolute eosinophil count compared to the SOC (Figure 3). More importantly, treatment with LEUKINE reduced ferritin and CRP levels while maintaining the absolute lymphocyte count in patients compared to the SOC (Figures 4-6). These may be markers of cytokine storm and potential toxicity, thus suggesting that LEUKINE can be safely administered to patients. Further evaluation of the immune response from D1 to D6 after LEUKINE treatment showed a significant increase in the number of SARS-CoV-2 antigen-specific HLD-DR+ CD38+ CD8+ T cells (Figure 8), as well as an increase in the number of activated IFN-γ and IL-2 secreting CD8+ T cells (Figure 9). However, treatment with LEUKINE did not result in a significant increase in IgG antibodies against SARS-CoV-2 spike (S1) and nucleocapsid (NCV) proteins, or IgA antibodies against the spike (S1) protein, as measured between days D1 and D6 (Figure 7).

[0125] Equivalents Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific embodiments described herein using experiments no more than routine experimentation. Such equivalents are intended to be covered within the scope of the following claims.

[0126] Reference All patents and documents referenced herein are incorporated herein by reference in their entirety.

[0127] Where used herein, all headings are solely for the structural purposes of this specification and are not intended to limit disclosure in any way. The content of any individual section may be equally applicable to all sections.

Claims

1. 1. A pharmaceutical composition for the treatment of acute respiratory syndrome in a patient with a coronavirus infection, comprising an effective amount of a composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), The pharmaceutical composition, wherein said treatment increases eosinophil counts in said patient.

2. The coronavirus is (i) betacoronaviruses (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43), and (ii) Alphacoronaviruses (e.g., HCoV-NL63 and HCoV-229E) The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is selected from the group consisting of:

3. 10. The pharmaceutical composition of claim 1, wherein the patient has one or more of fever, cough, shortness of breath, diarrhea, upper respiratory tract obstruction, lower respiratory tract obstruction, pneumonia, and acute respiratory distress syndrome, and / or the patient is hypoxic.

4. The pharmaceutical composition of claim 1 , wherein the treatment further comprises measuring the number of eosinophils in a biological sample from the patient.

5. 5. The pharmaceutical composition of claim 4, wherein the biological sample comprises blood, respiratory fluid, nasal secretions, saliva, sputum, or feces.

6. 6. The pharmaceutical composition of claim 5, wherein the respiratory fluid is from an oropharyngeal (OP) or nasopharyngeal (NP) swab or lavage fluid.

7. 10. The pharmaceutical composition of claim 1, wherein the treatment results in one or more of the following: an increase in HLA-DR+ CD38+ CD8+ T cells in said patient; a reduction in the patient's viral load compared to before treatment; a decrease in ferritin in said patient compared to before treatment, wherein said treatment causes a decrease in ferritin to less than 1000 ng / ml, or to less than 650 ng / ml; a decrease in C-reactive protein (CRP) compared to before treatment, wherein said treatment causes a decrease in CRP to less than 10 mg / L, or to less than 3 mg / L; improving oxygenation of said patient; preventing or alleviating the onset of acute respiratory distress syndrome (ARDS) in said patient; preventing or alleviating the transition from respiratory distress to cytokine imbalance in said patient; reversal or prevention of cytokine storm, wherein said cytokine storm is in the lungs or systemically and is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphocytosis; Reversal or prevention of overproduction of one or more inflammatory cytokines, wherein said inflammatory cytokines are one or more of IL-6, IL-1, IL-2, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

8. 2. The pharmaceutical composition of claim 1, wherein the GM-CSF has the amino acid sequence of any of SEQ ID NOs: 1 to 3, or a variant thereof having at least 90%, or 93%, or 95%, or 97%, or 98% amino acid sequence identity thereto.

9. 2. The pharmaceutical composition of claim 1, wherein the GM-CSF is one of molgramostim, sargramostim, and regramostim.

10. 10. The pharmaceutical composition of claim 1, wherein the GM-CSF is administered at a dose of 125 μg, or 150 μg, or 200 μg, or 250 μg, or 300 μg, or 350 μg.

11. the GM-CSF is sargramostim administered by inhalation at a dose of 125 μg twice daily; or The GM-CSF is sargramostim, administered intravenously at a dose of 125 μg; The pharmaceutical composition of claim 1.

12. 10. The pharmaceutical composition of claim 1, wherein the GM-CSF is administered by inhalation or to the lungs by an aerosol or nebulizer, the aerosol or nebulizer being selected from a liquid spray, a dry powder dispersion, and a metered dose administration.

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