Long-acting granulocyte-macrophage colony-stimulating factor

Abstract

Colony-stimulating factors (CSFs) refer to a family of four glycoproteins that control and regulate cell generation by the deposition of widely dispersed bone marrow cells. GM-CSF is a blood growth factor that controls the production, migration, proliferation, differentiation, and function of hematopoietic cells. The present disclosure generally relates to compositions and methods related to long-acting granulocyte-macrophage colony-stimulating factor (GM-CSF) with improved pharmacokinetics.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 329,039, filed April 8, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates, inter alia, to compositions and methods relating to long-acting granulocyte-macrophage colony-stimulating factor (GM-CSF) with improved pharmacokinetics.

[0003] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer-readable copy of the Sequence Listing (Filename: PNR-009PC 127114-5009-PC Sequence Listing.xml; Recorded Date: March 23, 2023; File Size: 20,480 bytes). [Background technology]

[0004] Colony-stimulating factors (CSFs) refer to a family of four glycoproteins that control and regulate cell generation from widely dispersed bone marrow cell deposits. These include granulocyte-macrophage CSF (GM-CSF), granulocyte-colony CSF (G-CSF), macrophage-colony CSF (M-CSF), and pluripotency-colony stimulating factor (IL-3). These lymphokines can induce precursor cells in the bone marrow to differentiate into specific types of mature blood cells. The specific types of mature blood cells that arise from precursor cells depend on the type of CSF present. See Metcalf D. Cancer Immunol Res. 2013, 1(6): 351-356.

[0005] GM-CSF is a blood growth factor that controls the production, migration, proliferation, differentiation, and function of hematopoietic cells. In response to inflammatory stimuli, GM-CSF is released by a variety of cell types, including T lymphocytes, macrophages, fibroblasts, and endothelial cells. GM-CSF then activates and enhances the production and survival of neutrophils, eosinophils, and macrophages. Natural GM-CSF is usually produced near the site of action, where it regulates the in vitro proliferation, differentiation, and survival of hematopoietic progenitor cells, but is present in the circulating blood at picomolar concentrations (10 -10 ~10 -12 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.

[0006] Human GM-CSF (hGM-CSF) is synthesized as a precursor protein of 144 amino acid residues including a 17 amino acid signal peptide. This precursor protein is processed to generate a mature protein of 127 amino acids with a predicted molecular weight of 14.4 kDa. The protein has two disulfide bonds that migrate as a broad band of 15-30 kDa due to glycosylation and sialylation. The N-linked glycans are Asn 27 and Asn 37GM-CSF is located at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and multiple potential sites for O-linked glycosylation exist at Ser5, Ser7, Ser9, and Ser10, although the extent of glycan structures at these sites has not been clearly determined. The glycosylation pattern of GM-CSF has been observed to affect its activity, receptor binding, immunogenicity, and half-life. See Lee F. et al. Proc Natl Acad Sci USA Biochem. 1985. 82: 360-4364; Miyatake S. et al. EMBO J. 1985. 4: 2561-2568. Cebon J et al. J Biol. Chem. 1991. 265, 4483-4491; Zhang Q et al. Proc. Natl. Acad. Sci. 2014, 2885-2890.

[0007] Recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) is FDA approved for the treatment of neutropenia, hematological disorders, and malignancies such as leukemia in combination with chemotherapy. In clinical practice, GM-CSF is used to treat post-chemotherapy neutropenia and aplastic anemia, significantly reducing the risk of infection associated with bone marrow transplantation. Its utility 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.

[0008] There are five classes of heterologous protein production platforms: bacteria, yeast, plants, insect cells, and mammalian cells, but more than 50% of currently marketed biopharmaceuticals are produced in mammalian cell lines. This is in part due to the inability of the remaining four classes to modify glycoproteins with human-like oligosaccharides. This is important because protein-bound glycans affect circulating half-life, tissue distribution, bioactivity, and immunogenicity.

[0009] The GM-CSF expression system affects the pharmacokinetic properties, bioactivity, and clinical toxicity of GM-CSF. In the clinic, GM-CSF is produced in Chinese hamster ovary cells (CHO-GM, regramostim), Escherichia coli (E.coli-GM, molgramostim), or yeast (Yeast-GM, sargramostim). See Dorr RT. Clin Therapeutics. 1993. 15(1):19-29; Walsh G. Nat Biotechnol. 2006, 24:769-776; Jacobs PP et al. Nat Protoc. 2009, 4:58-70; Jacobs PP et al. Microbial Cell Factories 2010, 9:93; Walsh G. Nat Biotechnol. 2018, 36(12): 1136-1145. During production of GM-CSF, four major GM-CSF isoforms / species exist: "hyperglycosylated" (approximately 50 kDa), N-glycosylated and N-+O-glycosylated (approximately 20 kDa), O-glycosylated (approximately 16 kDa), and non-glycosylated (approximately 15 kDa) species. Glycosylated GM-CSF is obtained by recombinant techniques using yeast (sargramostim) or Chinese hamster ovary (CHO) cell (ligramostim) technologies, which generate a complex mixture of glycoforms. GM-CSF from human cells is heterogeneously glycosylated as well, whereas GM-CSF produced in E. coli is not glycosylated. The heterogeneity of the glycans reflects the lack of specificity in post-translational glycosylation in CHO cells. See Dorr RT. Clinical Therapeutics / vol. 15, NO. 1, 1993; Zhang Q et al. PNAS. 2014. 111(8): 2885-2890.

[0010] N-glycosylation occurs when a sugar chain is added to the nitrogen of an asparagine (Asn) or arginine (Arg) side chain, whereas O-glycosylation occurs when a sugar chain is added to the hydroxyl oxygen side chain of a serine (Ser) or threonine (Thr) or tyrosine (Tyr) amino acid. Asparagine residues typically require the consensus sequence Asn-Xxx-Ser / Thr / Cys (where Xxx is any amino acid except proline) to be N-glycosylated. The rhuGM-CSF protein sequence of sargramostim contains two consensus sequences, Asn-Leu-Ser (NLS) and Asn-Glu-Thr (NET), at which the protein may be glycosylated. During the production of sargramostim, some GM-CSF species present in the fermenter contain Asn 27 and Asn 37 Both N-glycosylation sites of Thr are glycosylated. 39 (or Ser 39 or Cys 39 ) residue is Asn 27 Or Asn 37 Asparagine residue 37 is a key anchor that allows glycosylation at the ribosomal level. Species with N-oligosaccharides attached to asparagine residue 37 are called "hyperglycosylated" species (30-100 kDa) because these glycoforms contain hundreds of mannose residues. Hypermannosylation occurs only at N37.

[0011] The final product of sargramostim (LEUKINE) is composed of N- and O-glycosylated glycoforms in addition to the unglycosylated form. Thus, the glycoform profile of sargramostim is heterogeneous, but has been very consistent since its approval. Nearly half of the GM-CSF protein in sargramostim is unglycosylated, and just under one-third is fully N-glycosylated. Of the N-glycosylated species, most, if not all, are O-glycosylated. Highly branched N-linked oligosaccharide structures cover a significant portion of the molecule, including the H1 and H5 alpha helices, without interfering with the receptor binding site, the H6 alpha helix, and part of the C-terminal random coil. The carbohydrate moiety of recombinant GM-CSF varies depending on the cellular source, but glycosylation is not required for receptor binding or biological activity in vitro or in vivo. Yeast rhuGM-CSF and nonglycosylated E. coli-derived rhuGM-CSF exhibit similar specific activities as measured by both receptor affinity and specific cell growth activity. See Urdal and Park, 1988; Metcalf D. Cancer 1990: 65:2185-2195.

[0012] Endogenous proteins in the human body are degraded in vivo at different rates, which vary greatly depending on their function. For example, hemoglobin is present throughout the life of the red blood cell, and histones have half-lives measured in years, while other proteins such as ornithine decarboxylase (half-life 11 minutes) are degraded at a much faster rate. See Thomas E Creighton (1993). "Chapter 10 - Degradation". Proteins: Structures and Molecular Properties (2nd ed.). WH Freeman and Company. pp. 463-473. ISBN 0-7167-2317-4. In eukaryotes, most of the short-lived proteins in the intracellular space are degraded by the ubiquitin-proteasome pathway (UPP). See Kybuczkova L, et al. J Cell Mol Med. 2014. 18: 947-961. The UPP plays a central role in cellular homeostasis in the processing and degradation of proteins, such as those controlling cell cycle progression, apoptosis, and DNA repair. See Heinemeyer W, et al. J Biol Chem. 1997. 272: 25200-25209. Lysosomes also remove unwanted proteins from the cytoplasm and outside the cell. Extracellular material is taken up by endocytosis, and intracellular material is digested by autophagy. See Settembre C, et al. Nature Reviews Molecular Cell Biology. 2013.14 (5): 283-96. GM-CSF has been shown to have a half-life that can range from 50 to 85 minutes. See Cebon et al. Blood. 1988. 72: 1340-1347; Stagg et al 2004. The in vivo biological activity of natural and recombinant GM-CSF is highly dependent on its bioavailability. See Dorr RT. Clinical Therapeutics / vol. 15, NO. 1, 1993.

[0013] Glycosylation has been reported to stabilize various proteins. Terminal glycans carrying some charges (e.g., sialic acid) have been found to be important in regulating the circulating half-life of glycoproteins. There are also some reports that glycosylation is essential for maintaining the conformational stability of proteins against pH denaturation, for example in G-CSF (GRANOCYTE). See Nissen C. Eur J Cancer. 1994; 30A (Suppl 3):S12-14; Ono M. Eur J Cancer. 1994; 30A (Suppl 3):S7-11. Increased pH stability can also be artificially introduced into proteins, for example, by glycosylation of penicillin G acylase. The half-life of the glycosylated version of this protein was increased 13-fold at pH 3 and 7-fold at pH 10 compared to the non-glycosylated protein. See Masarova J, Biotechnol Appl Biochem. 2001; 34(Pt 2):127-133. It has also been shown that both the degree of glycosylation and the size of the glycan can increase the inactivation half-life of a protein, with increased glycan size providing a much greater degree of kinetic stabilization. See Sola RJ, Al-Azzam W, Griebenow K. Biotechnol Bioeng. 2006; 94(6):1072-1079; Sola RJ, Cell Mol Life Sci. 2007; 64(16):2133-2152; Sola RJ et al. J Pharm Sci. 2009. 98(4): 1223-1245.

[0014] The bioavailability of therapeutic proteins (biotherapeutics or biologics) in humans may also be affected by the route of administration of the drug. See Dorr RT, Clinical Therapeutics / vol. 15, NO. 1, 1993. After intravenous (IV) administration, the therapeutic protein is nearly fully available, but may subsequently be degraded over time by proteases present in the plasma. In some cases, subcutaneous (SC) administration may extend exposure to proteins with short elimination half-lives by maintaining high plasma concentrations for extended periods of time, which may improve tolerability compared to IV administration. Thus, absorption of biotherapeutics after SC administration may be affected by their fate within the extracellular matrix (ECM), including the possibility of incomplete bioavailability. See Hale G, et al. Blood. 2004.104(4):948-955. Different isoforms present in sargramostim may be transported into the systemic circulation by different routes, due to their different molecular sizes.

[0015] The GM-CSF expression system can affect pharmacokinetic parameters, with the degree of glycosylation influencing the half-life, distribution, and excretion of GM-CSF. The relationship between the degree of GM-CSF glycosylation and its distribution, clearance, and activity has been reported using a rat model. The specific activity of GM-CSF measured in vitro was found to be significantly reduced in the largest, most fully glycosylated protein form compared to the smaller, less glycosylated molecule. The effective half-life of GM-CSF in the bloodstream of rats after a single intravenous bolus injection was shown to be significantly increased by the addition of N-linked carbohydrate. The clearance of GM-CSF in rats follows biphasic kinetics, with the first phase, the c-phase, being prolonged by carbohydrate modification. See Donohue RE, et al. Cold Spring Harbor Labs. 1986. 51: 685-692, and Dorr RT Clinical Therapeutics. 1993. 15(1): 19-29. Another way to influence pharmacokinetic parameters is to conjugate targeting proteins such as GM-CSF to albumin, which takes advantage of the long half-life and targeting ability of albumin to increase the biostability of the targeting protein. See Chuang YM et al. Cell Mol Immunol. 2020.

[0016] Anti-serum albumin domain antibodies have also been shown to extend the half-life of short-lived drugs. See Holt LJ et al. Protein Eng Des Sel. 2008. 21(5):283-8. However, the large size of conventional antibodies can hinder the delivery of target proteins or drugs to cells in vivo. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed exclusively of heavy chains whose target recognition modules consist of a single variable domain (VHH or Nb). Advantages of nanobodies include small size, high solubility, high stability, and excellent tissue penetration in vivo. See Bannas P et al. Front Immunol. 2017. The in vivo half-life of molecules, e.g. antitumor nanobodies, can be extended by fusing one or more antitumor nanobodies to an albumin-specific nanobody. See Roovers RC et al. Int J Cancer. 2011. 129(8):2013-24; Zhu Y et al. Sci Rep (2017) 7(1):2602; Bannas P et al. Front Immunol. 2017. It stands to reason that by extending the half-life and circulation time of GM-CSF-based therapies, we could reduce the dose of drug administered to patients and simplify the treatment regimen. This could have a positive impact on the quality of patient care, patient compliance, and reduced healthcare costs.

[0017] There is a need for GM-CSF molecules with improved pharmacokinetics, having an extended half-life and extended circulation time. Summary of the Invention

[0018] Thus, the present disclosure relates to long-acting forms of GM-CSF that provide improved pharmacokinetics and pharmacodynamics, particularly as compared to wild-type GM-CSF or sargramostim.

[0019] In some embodiments, a fusion or chimeric protein is provided comprising a recombinant human GM-CSF protein, a linker, and a humanized single domain antibody.

[0020] In some embodiments, the recombinant human GM-CSF protein of the compositions of the invention comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:1 or SEQ ID NO:2, optionally with a substitution or deletion at position N37 or a corresponding position, e.g., the amino acid at position N37 or a corresponding position may be substituted with a polar, neutrally charged hydrophilic amino acid, such as glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

[0021] In some embodiments, the humanized single domain antibody of the composition of the invention comprises a heavy chain having three CDRs, for example as defined by the IMGT nomenclature or the Kabat nomenclature. IMGT nomenclature: CDR-H1: GETLDYYA (SEQ ID NO: 12) CDR-H2: IASSGGT (SEQ ID NO: 13) CDR-H3: AAAVLECRTVVRGYDY (SEQ ID NO: 14) Kabat CDR definition: CDR-H1: ETLDYYAIG (SEQ ID NO: 15) CDR-H2: IASSGGSTNYADSVKG (SEQ ID NO: 16) CDR-H3: AVLECRTVVRGYDY (SEQ ID NO: 17).

[0022] In some embodiments, the humanized single domain antibody of the composition of the invention comprises a heavy chain with three CDRs of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, or a variant with one or more substitutions or deletions (e.g., one, two, three, or four). In some embodiments, the humanized single domain antibody of the composition of the invention comprises a heavy chain with three CDRs of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or a variant with one or more substitutions or deletions (e.g., one, two, three, or four). In some embodiments, the humanized single domain antibody of the composition comprises an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to SEQ ID NO: 3, with substitutions or deletions within the non-human llama antibody framework to allow humanization of the non-human antibody. CDRs are defined as per IMGT nomenclature. CDRs can also be defined by the Kabat nomenclature. IMGT nomenclature: CDR-H1: GETLDYYA (SEQ ID NO: 12) CDR-H2: IASSGGT (SEQ ID NO: 13) CDR-H3: AAAVLECRTVVRGYDY (SEQ ID NO: 14) Kabat CDR definition: CDR-H1: ETLDYYAIG (SEQ ID NO: 15) CDR-H2: IASSGGSTNYADSVKG (SEQ ID NO: 16) CDR-H3: AVLECRTVVRGYDY (SEQ ID NO: 17)

[0023] In some embodiments, in a fusion protein or chimeric protein, a protein comprising recombinant human GM-CSF protein is fused to a humanized single domain antibody via a linker.

[0024] In some embodiments, a composition of the invention comprises multiple molecular forms, e.g., unglycosylated, O-glycosylated, N-glycosylated, and / or N+O glycosylated forms. In some embodiments, a fusion protein or chimeric protein of the invention is substantially free of hyperglycosylated forms, e.g., hypermannosylated forms.

[0025] In some embodiments, nucleic acid molecules (e.g., codon optimized sequences) encoding the fusion or chimeric proteins of the invention are also provided. In some embodiments, human host cells (optionally Chinese Hamster Ovary (CHO) cells) expressing the nucleic acid molecules encoding the fusion or chimeric proteins of the invention are also provided. In some embodiments, pharmaceutical compositions comprising the fusion or chimeric proteins of the invention and a pharma- ceutically acceptable excipient or carrier are also provided.

[0026] In some embodiments, methods are provided for treating a patient or subject undergoing or having undergone cancer treatment, or a patient or subject undergoing or having undergone a bone marrow transplant, or a patient or subject acutely exposed to a myelosuppressive amount of radiation (e.g., Acute Radiation Sickness or Acute Radiation Syndrome (ARS)), and / or a patient or subject with Radiation Combined Injury (RCI), comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition described herein.

[0027] In some embodiments, a method of treating a neurodegenerative disease (such as, but not limited to, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), dementia with Lewy bodies, Parkinson's dementia, epilepsy, stroke, Huntington's disease, cerebral hypoxia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and peripheral neuropathy) is provided, wherein a subject in need of treatment is treated with a fusion protein or chimeric protein described herein or a pharmaceutical composition thereof.

[0028] In some embodiments, a method of treating a fungal infection (e.g., an invasive fungal infection, optionally selected from, but not limited to, an infection due to candidiasis and / or mucormycosis) is provided, wherein a subject in need of treatment is treated with a fusion or chimeric protein or pharmaceutical composition thereof described herein.

[0029] In some embodiments, a method of treating an autoimmune disease, optionally selected from Crohn's disease and / or ulcerative colitis (UC), irritable bowel disease (IBD) and / or other GI inflammatory diseases, is provided, wherein a patient or subject in need of treatment is treated with a fusion or chimeric protein or pharmaceutical composition thereof as described herein.

[0030] In some embodiments, the fusion protein or chimeric protein of the present invention is functionally similar to sargramostim. In some embodiments, the fusion protein or chimeric protein has improved pharmacokinetics compared to recombinant human GM-CSF. In some embodiments, the fusion protein or chimeric protein has improved half-life compared to recombinant human GM-CSF. In some embodiments, the fusion protein or chimeric protein has improved pharmacodynamics compared to recombinant human GM-CSF. [Brief description of the drawings]

[0031] [Figure 1A] FIG. 1A illustrates the structure of heavy chain IgG (hcIgG).

[0032] [Figure 1B] Figure 1B shows the yeast surface display system used to screen the antigen-binding scaffold library. The VHH is fused via its C-terminus to the N-terminus of Aga2p. Surface expression can be detected using a fluorescently labeled antibody that binds to the Myc tag.

[0033] [Figure 1C] Figure 1C is a graph showing the half-life extension of Fab fragments promoted by VHH in a mouse model. The different lines on the graph represent the different constructs. Bi-340-352 is an anti-albumin VHH half-life extender as a fusion partner with the Fab fragment. Bi-340-356 is an unrelated VHH fusion with the same Fab fragment that does not show half-life extension.

[0034] [Diagram 2] Figure 2 shows four constructs with or without secretory leader sequences that were cloned and transiently expressed in CHO cells. The secretory leader sequence was the same in all four constructs. The four constructs include VHH-G4Sx4 linker-GMCSF, VHH-hinge linker-GMCSF, GMCSF-hinge linker-VHH, and GM-CSF-G4Sx4 linker-VHH. Also shown in the figure is VHH-G4Ax4 linker-GM-CSF with or without secretory leader sequence.

[0035] [Figure 3A]3A,B are graphs showing the results of TF-1 assays with and without human serum albumin (HSA) for various fusion or chimeric proteins, CHO-produced GM-CSF (ATUM CHO N37Q), and LEUKINE / QCRS-GMCSF (displayed as separate graph lines). BDS LEUKINE / QCRS GMCSF refers to wild-type GM-CSF (SEQ ID NO: 2) without any of the amino acid substitutions T39A or N37Q in the sequence. CHO N37Q is the CHO-produced GMCSF variant used in the VHH construct. HG-GS-H is the GMCSF-G4Sx4-VHH and HH-GS-G is the VHH G4Sx4-GMCSF molecule. The TF1 cell-based assay was repeated with or without the addition of HSA at 40 μg / ml (physiological concentration in blood) to the cell assay. [Figure 3B] Figure 3A,B are graphs showing the results of TF-1 assays with and without human serum albumin (HSA) for various fusion or chimeric proteins, CHO-produced GM-CSF (ATUM CHO N37Q) and LEUKINE / QCRS-GMCSF (displayed as separate graph lines). BDS LEUKINE / QCRS GMCSF refers to wild-type GM-CSF (SEQ ID NO: 2) without any of the amino acid substitutions T39A or N37Q in the sequence. CHO N37Q is the CHO-produced GMCSF variant used in the VHH construct. HG-GS-H is the GMCSF-G4Sx4-VHH and HH-GS-G is the VHH G4Sx4-GMCSF molecule. The TF1 cell-based assay was repeated with or without the addition of HSA at 40 μg / ml (physiological concentration in blood) to the cell assay.

[0036] [Figure 4A]Figure 4A shows an example of a spectrograph used to confirm the heterogeneity of N-terminal truncation and glycosylation of the VHH-G4Sx4-GMCSF variant (VHH-G4Sx4-GMCSF*). High-resolution liquid chromatography mass spectrometry (LC-MS) (Vanquish UPLC Q Exactive, Thermo) was used to determine the intact molecular weight and glycan profile. The figure shows the mass spectrometric glycan analysis, specifically the glycan occupancy of six stable CHO clones producing VHH-G4A GMCSF*. 100 μg of sample was diluted to 2 mg / mL in deionized water and injected into the LC-MS. The data was analyzed and deconvoluted with BioPharmaFinder software to obtain the intact protein molecular weight. Mass spectrometry also showed that the known glycosylation sites of GMCSF, Ser7, Ser10, and N27, were also glycosylated in all VHH-G4Sx4-GMCSF variants, similar to the CHO-produced GMCSF* used in the fusion protein.

[0037] [Figure 4B] Figure 4B is a graph showing the results of the TF-1 assay. VHH-G4A GMCSF* generated from six stable CHO clones were measured in the TF-1 cell-based assay.

[0038] [Figure 5A]5A-C are graphs showing surface plasmon resonance (SPR) evaluation of VHH binding to three albumins including human, mouse, and rhesus. To evaluate binding, VHHs were cloned onto human IgG1 Fc and hinge for ease of purification and analysis. Binding affinity of VHH-Fc constructs was measured by BLI using Octet384 (Pall Life Sciences). VHH-Fc constructs were loaded onto AHC sensors and then exposed to monomeric targets human albumin, mouse albumin, or rhesus albumin in solution, and binding and dissociation were measured. For pH evaluation, binding reaction and dissociation buffers were at the indicated pH. Data acquisition was performed at 30°C. [Figure 5B] 5A-C are graphs showing surface plasmon resonance (SPR) evaluation of VHH binding to three albumins including human, mouse, and rhesus. To evaluate binding, VHHs were cloned onto human IgG1 Fc and hinge for ease of purification and analysis. Binding affinity of VHH-Fc constructs was measured by BLI using Octet384 (Pall Life Sciences). VHH-Fc constructs were loaded onto AHC sensors and then exposed to monomeric targets human albumin, mouse albumin, or rhesus albumin in solution, and binding and dissociation were measured. For pH evaluation, binding reaction and dissociation buffers were at the indicated pH. Data acquisition was performed at 30°C. [Figure 5C]5A-C are graphs showing surface plasmon resonance (SPR) evaluation of VHH binding to three albumins including human, mouse, and rhesus. To evaluate binding, VHHs were cloned onto human IgG1 Fc and hinge for ease of purification and analysis. Binding affinity of VHH-Fc constructs was measured by BLI using Octet384 (Pall Life Sciences). VHH-Fc constructs were loaded onto AHC sensors and then exposed to monomeric targets human albumin, mouse albumin, or rhesus albumin in solution, and binding and dissociation were measured. For pH evaluation, binding reaction and dissociation buffers were at the indicated pH. Data acquisition was performed at 30°C.

[0039] [Figure 6A] Figures 6A-D are graphs showing surface plasmon resonance (SPR) assessment of VHH binding to human albumin at various pHs at 30°C. The graphs show response units (target binding to VHH-Fc on the chip sensor) on the Y-axis and time on the X-axis. The data (time vs. response units) were analyzed using ForteBio data analysis software. [Figure 6B] Figures 6A-D are graphs showing surface plasmon resonance (SPR) assessment of VHH binding to human albumin at various pHs at 30°C. The graphs show response units (target binding to VHH-Fc on the chip sensor) on the Y-axis and time on the X-axis. The data (time vs. response units) were analyzed using ForteBio data analysis software. [Figure 6C] Figures 6A-D are graphs showing surface plasmon resonance (SPR) assessment of VHH binding to human albumin at various pHs at 30°C. The graphs show response units (target binding to VHH-Fc on the chip sensor) on the Y-axis and time on the X-axis. The data (time vs. response units) were analyzed using ForteBio data analysis software. [Figure 6D]Figures 6A-D are graphs showing surface plasmon resonance (SPR) assessment of VHH binding to human albumin at various pHs at 30°C. The graphs show response units (target binding to VHH-Fc on the chip sensor) on the Y-axis and time on the X-axis. The data (time vs. response units) were analyzed using ForteBio data analysis software.

[0040] [Figure 7A] 7A-C show graphs of the half-life (t1 / 2) or pharmacokinetics of the fusion or chimeric protein, VHH-GA-GMCSF*, compared to CHO-produced GM-CSF and LEUKINE after intravenous injection of 10 μg / kg of the indicated form of GMCSF. One rhesus monkey was intravenously injected with 10 μg / kg of the indicated form of GMCSF. Blood was collected 12 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after injection to measure cell content and concentration of construct in blood by construct-specific ELISA. VHH-GA-GMCSF* shows a t1 / 2 approximately 17-fold greater than the CHO-produced form of GMCSF used in the LEUKINE or VHH-GA-GMCSF constructs. The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. The yeast control is LEUKINE. [Figure 7B]7A-C show graphs of the half-life (t1 / 2) or pharmacokinetics of the fusion or chimeric protein, VHH-GA-GMCSF*, compared to CHO-produced GM-CSF and LEUKINE after intravenous injection of 10 μg / kg of the indicated form of GMCSF. One rhesus monkey was intravenously injected with 10 μg / kg of the indicated form of GMCSF. Blood was collected 12 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after injection to measure cell content and concentration of construct in blood by construct-specific ELISA. VHH-GA-GMCSF* shows a t1 / 2 approximately 17-fold greater than the CHO-produced form of GMCSF used in the LEUKINE or VHH-GA-GMCSF constructs. The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. The yeast control is LEUKINE. [Figure 7C] 7A-C show graphs of the half-life (t1 / 2) or pharmacokinetics of the fusion or chimeric protein, VHH-GA-GMCSF*, compared to CHO-produced GM-CSF and LEUKINE after intravenous injection of 10 μg / kg of the indicated form of GMCSF. One rhesus monkey was intravenously injected with 10 μg / kg of the indicated form of GMCSF. Blood was collected 12 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after injection to measure cell content and concentration of construct in blood by construct-specific ELISA. VHH-GA-GMCSF* shows a t1 / 2 approximately 17-fold greater than the CHO-produced form of GMCSF used in the LEUKINE or VHH-GA-GMCSF constructs. The CHO control is the GMCSF variants (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. The yeast control is LEUKINE.

[0041] [Figure 8A] Figures 8A-E show graphs of neutrophil, monocyte, eosinophil, total white blood cell, and platelet counts measured after intravenous (IV) injection of fusion or chimeric protein constructs compared to CHO and LEUKINE (yeast-cultured GM-CSF). The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. [Figure 8B] Figures 8A-E show graphs of neutrophil, monocyte, eosinophil, total white blood cell, and platelet counts measured after intravenous (IV) injection of fusion or chimeric protein constructs compared to CHO and LEUKINE (yeast-cultured GM-CSF). The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. [Figure 8C] Figures 8A-E show graphs of neutrophil, monocyte, eosinophil, total white blood cell, and platelet counts measured after intravenous (IV) injection of fusion or chimeric protein constructs compared to CHO and LEUKINE (yeast-cultured GM-CSF). The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. [Figure 8D] Figures 8A-E show graphs of neutrophil, monocyte, eosinophil, total white blood cell, and platelet counts measured after intravenous (IV) injection of fusion or chimeric protein constructs compared to CHO and LEUKINE (yeast-cultured GM-CSF). The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. [Figure 8E]Figures 8A-E show graphs of neutrophil, monocyte, eosinophil, total white blood cell, and platelet counts measured after intravenous (IV) injection of fusion or chimeric protein constructs compared to CHO and LEUKINE (yeast-cultured GM-CSF). The CHO control is the GMCSF variant (GMCSF R23L and N37Q) used in the VHH constructs made in CHO cells to maintain a similar glycosylation pattern. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present disclosure is based, in part, on a fusion or chimeric protein comprising recombinant human GM-CSF protein, a linker, and a humanized single domain antibody, which composition produces functional GM-CSF with improved half-life, pharmacokinetics, and pharmacodynamics.

[0043] Composition of GM-CSF In one aspect, modified fusion or chimeric proteins are provided.

[0044] In some embodiments, the modified GM-CSF used in the practice of the present disclosure includes any pharma- ceutical safe and effective GM-CSF, or any derivative thereof that has the biological activity of GM-CSF, optionally having the substitutions and / or deletions of the invention.

[0045] In one embodiment, the modified GM-CSF used in the practice of the methods of the invention is derived from recombinant human GM-CSF (rhuGM-CSF), such as sargramostim (LEUKINE). Sargramostim is a biosynthetic, yeast-derived, recombinant human GM-CSF, which has a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF in having a leucine instead of arginine at position 23. Other natural and synthetic GM-CSFs, and derivatives thereof that have the biological activity of natural human GM-CSF, may be similarly useful in the practice of the present disclosure.

[0046] In some embodiments, the recombinant human GM-CSF molecules of the invention are glycosylated. In some embodiments, the recombinant human GM-CSF molecules of the invention contain one or more substitutions and / or deletions that affect the glycosylation of GM-CSF.

[0047] Without wishing to be bound by theory, it is believed that the degree of glycosylation of biosynthetic GM-CSF affects the half-life, distribution, and excretion (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).

[0048] In one aspect, there is provided a recombinant human GM-CSF protein comprising an amino acid sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity, or 100% identity, to SEQ ID NO: 1 or SEQ ID NO: 2. In one aspect, there is provided a recombinant human GM-CSF protein comprising an amino acid sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity, or 100% identity, to SEQ ID NO: 1 or SEQ ID NO: 2, and with a substitution or deletion at position N37 or a position corresponding thereto.

[0049] In some embodiments, the amino acid at or corresponding to position N37 of recombinant human GM-CSF is a polar, neutrally charged hydrophilic amino acid. In some embodiments, the polar, neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, the polar, neutrally charged hydrophilic amino acid is glutamine (Q). In some embodiments, the polar, neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S), and threonine (T).

[0050] In some embodiments, a recombinant human GM-CSF protein is provided that comprises an amino acid sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO:1 or SEQ ID NO:2, and having one or more of the following N37 deletions: N37Q, N37S, N37T, N37P, and N37C.

[0051] In some embodiments, the recombinant human GM-CSF has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a variant having about 90%, about 93%, about 95%, about 97%, or about 98% identity thereto, optionally with substitutions and / or deletions of the invention.

[0052] In some embodiments, the GM-CSF is any of molgramostim, sargramostim, and regramostim, optionally with substitutions and / or deletions of the invention.

[0053] Without wishing to be bound by theory, the core of hGM-CSF is composed of four helices packed at angles. Crystal structure and mutagenesis analysis of rhGM-CSF (Rozwarski DA et al., Proteins 26:304-13, 1996) showed that in addition to the nonpolar side chains of the protein core, 10 buried hydrogen-bonding residues are involved in intramolecular hydrogen bonding to main-chain atoms, which are better conserved than residues hydrogen-bonding to other side-chain atoms; 24 solvation sites were observed at equivalent positions in the asymmetric units of the two molecules, the strongest of which are located in clefts between secondary structure elements. Two surface clusters of hydrophobic side chains are located near the predicted receptor-binding region. Mutagenesis of residues in the helix A / helix C face confirmed the importance of specific Glu, Gly, and Gin residues. Thus, these residues are not replaced in functional substitution variants of hGM-CSF for use in this disclosure, and these helices are retained in functional fragment or deletion variants of hGM-CSF for use in this disclosure.

[0054] Additionally, in some embodiments, one of skill in the art can refer to UniProtKB entry P04141 for structural information to obtain information about the identity of variants.

[0055] The N-terminal helix of hGM-CSF controls the high affinity binding to its receptor (Shanafelt AB et al., EMBO J 10:4105-12, 1991). Transduction of the biological effects of GM-CSF requires interaction with at least two cell surface receptor components, one of which is shared with the cytokine IL-5. In the above studies, we identified the receptor binding determinants of GM-CSF by placing unique receptor binding domains on a series of human-mouse hybrid GM-CSF cytokines. The interaction of GM-CSF with the shared subunit of their high affinity receptor complex was controlled by a small portion of the peptide chain. The presence of a few key residues in the N-terminal α-helix was sufficient to confer specificity to the interaction.

[0056] Thus, in some embodiments, this information may inform one of skill in the art regarding permissible variations in an amino acid sequence.

[0057] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0058] "Conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature 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 influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0059] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid that is listed in the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to break alpha helices.

[0060] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0061] In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine; 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; sarcosme; citrulline; homocitrulline; cysteic acid; t-butylglycine; t-butylalanine; phenylglycine; cyclohexylalanine; β-alanine; fluoroamino acids; artificially designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids; and common amino acid analogs).

[0062] Amino acid sequence modifications can be accomplished using techniques known in the art, such as site-directed mutagenesis or PCR-based mutagenesis, as 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.

[0063] Glycoform In some embodiments, the fusion or chimeric protein of the invention comprises a GM-CSF molecule that comprises multiple molecular forms, which in some embodiments are selected from an unglycosylated form, an O-glycosylated form, an N-glycosylated form, and an N+O glycosylated form.

[0064] In some embodiments, the fusion protein or chimeric protein is substantially free of hyperglycosylated forms, e.g., hypermannosylated forms. In some embodiments, the recombinant human GM-CSF has about 10% or less hypermannosylated forms after purification, about 5% or less hypermannosylated forms after purification, about 3% or less hypermannosylated forms after purification, about 2% or less hypermannosylated forms after purification, or about 1% or less hypermannosylated forms after purification.

[0065] In some embodiments, the fusion protein or chimeric protein has less hypermannosylated forms than wild-type human GM-CSF and / or sargramostim when expressed and purified by the same methods. In some embodiments, the recombinant human GM-CSF has about 100%, or about 90%, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% less hypermannosylated forms than wild-type human GM-CSF and / or sargramostim when expressed and purified by the same methods. In some embodiments, the amount of hypermannosylated forms can be detected by methods known in the art, including, but not limited to, chromatographic methods (e.g., size separation (e.g., via molecular weight and / or retention time on a column and / or fluorescent labeling (e.g., using 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 2-aminopyridine (2-AP))), anion exchange chromatography, etc.), mass spectrometry, SDS-PAGE / staining (e.g., gel staining procedures based on the Periodic Acid Schiff (PAS) reaction), affinity-based methods such as the use of carbohydrate-binding proteins (e.g., lectins), enzyme-based methods, antibody-based methods, release assays (e.g., enzymatic cleavage or chemical removal of carbohydrate moieties, or chemical derivatization), capillary electrophoresis, and Eastern blots, etc.).

[0066] In some embodiments, the fusion or chimeric proteins of the present invention are amenable to less complicated purification procedures than those used for sargramostim.

[0067] In some embodiments, the fusion or chimeric GM-CSF proteins of the invention are resolved into three peaks when quantified by reversed-phase high performance liquid chromatography (RP-HPLC). In some embodiments, the compositions of the invention are resolved, for example by HPLC, into three forms (an unglycosylated form, an O-linked glycoform, and an O+N-linked glycoform) and lack the hyperglycosylated form.

[0068] In some embodiments, the fusion or chimeric GM-CSF proteins of the invention can be purified free of hyperglycosylated species without the need for one or more organic solvents (e.g., but not limited to, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid, and / or N-propanol) and / or reverse-phase C4 HPLC columns used for the purposes of purification and / or removal of the hyperglycosylated peaks (unglycosylated, O-linked glycoforms, and O+N-linked glycoforms).

[0069] In some embodiments, recombinant human GM-CSF of the present invention comprises multiple species (eg, glycoforms).

[0070] In some embodiments, the fusion or chimeric proteins produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, are monomeric with heterologous glycoforms, including both glycosylated and non-glycosylated isoforms.

[0071] Humanized single domain antibodies (VHH) In some embodiments, the humanized single domain antibody of the composition of the invention comprises an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to SEQ ID NO:3, with substitutions or deletions within the non-human llama antibody framework to allow humanization of the non-human antibody. CDRs are defined by the IMGT nomenclature. CDRs can also be defined by the Kabat nomenclature.

[0072] In some embodiments, the humanized single domain antibody of the composition comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO:3, and / or comprises three CDRs selected from (i) SEQ ID NOs:12-14 or a variant thereof, or (ii) SEQ ID NOs:15-17 or a variant thereof.

[0073] In some embodiments, the humanized single domain antibodies (VHH) of the compositions bind to human, mouse, and rhesus serum albumin. In some embodiments, the humanized single domain antibodies (VHH) of the compositions bind to and / or are specific for human serum albumin (HSA). In some embodiments, the compositions of the present disclosure bind to HSA, where HSA is present in human plasma.

[0074] In some embodiments, the composition does not substantially compete with the binding of HSA to the neonatal Fc receptor (FcRn).

[0075] In some embodiments, the composition binds to Protein A.

[0076] In some embodiments, when the fusion protein or chimeric protein of the present invention is soluble, the protein binding activity may be expressed as a dissociation constant (KD). Optionally, when the fusion protein or chimeric protein is a membrane antigen, the antigen binding activity may be expressed as an apparent dissociation constant (apparent KD).

[0077] In some embodiments, the dissociation constant KD and the apparent dissociation constant (apparent KD) can be determined by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scatchard plot, or FACS.

[0078] In some embodiments, the disclosure provides compositions having a KD of at least about 50 nM, or at least about 45 nM, or at least about 40 nM, or at least about 35 nM, or at least about 30 nM, or at least about 25 nM, or at least about 20 nM, or at least about 15 nM, or at least about 10 nM, or at least about 5 nM. In some embodiments, the disclosure provides compositions having an apparent KD of at least about 100 pM, or at least about 90 pM, or at least about 80 pM, or at least about 70 pM, or at least about 60 pM, or at least about 50 pM, or at least about 40 pM, or at least about 30 pM, or at least about 20 pM, or at least about 10 pM.

[0079] In some embodiments, the binding activity of a composition may be measured under different conditions. In preferred embodiments, the measurement conditions are constant.

[0080] Linker In some embodiments, a fusion protein or chimeric protein, a protein comprising a recombinant human GM-CSF protein, is linked and / or fused to a humanized single domain antibody via a linker.

[0081] In some embodiments, the linker of the composition is a flexible linker. In some embodiments, the flexible linker of the composition is substantially composed of glycine and serine residues. In some embodiments, the flexible linker of the composition comprises at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60 amino acid residues.

[0082] In one embodiment, the flexible linker of the composition of the invention is (Gly4Ser) nを where n is from about 1 to about 12.

[0083] In one embodiment, the flexible linker of the composition of the invention is (Gly4Ala).nを where n is from about 1 to about 12.

[0084] In one embodiment, the flexible linker of the composition is a hinge-CH2-CH3 Fc domain. In one embodiment, the hinge-CH2-CH3 Fc domain of the composition is derived from human IgG. In some embodiments, the hinge-CH2-CH3 Fc domain of the composition is derived from human IgG1, IgG2, or IgG4.

[0085] In some embodiments, the linker of the composition comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the linker of the composition comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO: 6. In some embodiments, the linker of the composition comprises an amino acid sequence having SEQ ID NO: 4 or SEQ ID NO: 5 or a variant thereof (e.g., having about 5, or about 4, or about 3, or about 2, or about 1 substitution or deletion).

[0086] In some embodiments, the VHH domain of the composition of the invention is placed at the N-terminus of the fusion protein or chimeric protein.

[0087] In some embodiments, the recombinant GM-CSF of the compositions of the invention is positioned at the N-terminus of the fusion protein or chimeric protein.

[0088] In some embodiments, compositions of the invention that include recombinant GM-CSF at the N-terminus of a fusion protein or chimeric protein have increased yields compared to compositions that include recombinant GM-CSF at the C-terminus.

[0089] Functional properties of recombinant GM-CSF, including fusion or chimeric proteins In some embodiments, the fusion or chimeric proteins of the invention, optionally having substitutions and / or deletions of the invention, are functionally similar to wild-type human GM-CSF and / or sargramostim (e.g., the difference in one or more functional parameters is about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or about 5-fold or less, or about 4-fold or less, or about 3-fold or less, or about 2-fold or less of the assayed functional parameter). In some embodiments, said functional parameters of GM-CSF can be detected by assays known in the art, such as, for example, but not limited to, proliferation assays using cells such as TF-1 cell line, primary bone marrow cells, biochemical assays such as ILITE (EAGLE) GM-CSF (luciferase under the control of the GM-CSF promoter); cell survival assays such as bone marrow cell survival assays; cell differentiation assays; and co-culture experiments.

[0090] In some embodiments, the fusion proteins or chimeric proteins of the invention, optionally having substitutions and / or deletions of the invention, bind to and / or activate the granulocyte macrophage colony stimulating factor receptor (GM-CSF-R-α or CSF2R). In some embodiments, the recombinant human GM-CSF molecules of the invention, optionally having substitutions and / or deletions of the invention, bind to and / or activate the granulocyte macrophage colony stimulating factor receptor (GM-CSF-R-α or CSF2R) with affinity, potency, and / or bioactivity equivalent to wild-type human GM-CSF and / or sargramostim (e.g., about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or about 5-fold or less, or about 4-fold or less, or about 3-fold or less, or about 2-fold or less). Assays for GM-CSF binding and activation are known in the art. Non-limiting examples of such assays include, for example, radioactive or non-radioactive ligand assays (e.g., immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), Western blot, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and radioimmunoassay (RIA)). Binding kinetics can also be assessed by standard assays known in the art, such as Biacore analysis. Whole cell ligand binding assays and cell-free assay systems using soluble GM-CSF receptor alpha (sGMRa) can also be used. Other types of assays that can be used include receptor binding assays, saturation binding assays, or competitive binding assays using radioiodinated GM-CSF, as well as cell proliferation assays.

[0091] In some embodiments, the fusion proteins or chimeric proteins of the invention can be assayed using one or more cell-based activity bioassays, such as using a GM-CSF-dependent human cell line proliferation assay, such as TF-1, M-07e, HU-3, M-MOK, MB-02, GM / SO, F-36P, GF-D8, ELF-153, AML-193, MUTZ-3, OCI-AML5, OCI-AML6, OCI-AML1, SKNO-1, UCSD-AML1, and UT-7.

[0092] In some embodiments, the potency of the fusion or chimeric proteins of the invention is measured using a bioassay with TF-1 cells, a human erythroblastic leukemia cell line that proliferates in response to GM-CSF. Details of this assay are known in the art. For example, reference standards, controls, and test samples are serially diluted in triplicate in assay medium and added to three separate 96-well plates. TF-1 cells in suspension are then added and the mixture is incubated at 37° C. for about 69.5-72 hours. After addition of a fluorescent dye (e.g., ALAMARBLUE), the plate is incubated at 37° C. for about 6.6-8 hours. The proliferation of TF-1 cells is then measured with a fluorescent microplate reader.

[0093] In some embodiments, the fusion or chimeric protein of the invention has approximately the same specific activity as recombinant human GM-CSF lacking the mutation (eg, when assayed using a bioassay with TF-1 cells).

[0094] In some embodiments, the functionality of a fusion protein or chimeric protein of the invention produced in a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, is equivalent to recombinant human GM-CSF produced in a yeast cell (e.g., less than about 90%, or less than about 80%, or less than about 70%, or less than about 60%, or less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 10-fold, or less than about 9-fold, or less than about 8-fold, or less than about 7-fold, or less than about 6-fold, or less than about 5-fold, or less than about 4-fold, or less than about 3-fold, or less than about 2-fold).

[0095] In some embodiments, the fusion or chimeric proteins of the invention have improved pharmacokinetics and pharmacodynamics compared to sargramostim or wild-type GM-CSF, hi some embodiments, the fusion or chimeric proteins of the invention have an extended half-life (e.g., differs in one or more functional parameters by about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, or about 5% or less, or about 5-fold or less, or about 4-fold or less, or about 3-fold or less, or about 2-fold or less) compared to sargramostim or wild-type GM-CSF.

[0096] In some embodiments, the GM-CSF-R-α that binds and / or activates is expressed on the surface of a cell. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the hematopoietic progenitor cell is an immune cell. In some embodiments, the hematopoietic progenitor cell is irradiated.

[0097] In some embodiments, the immunogenicity of the fusion or chimeric proteins of the invention, optionally having the substitutions and / or deletions of the invention, is comparable to wild-type human GM-CSF and / or sargramostim (e.g., about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or about 5-fold or less, or about 4-fold or less, or about 3-fold or less, or about 2-fold or less difference in one or more functional parameters). In some embodiments, immunogenicity is assayed using methods known in the art. Non-limiting examples include detection of one or more anti-GM-CSF binding antibodies, as assessed by screening assays such as direct or indirect or bridging ELISA, electrochemiluminescence, bead-based chemiluminescence assays, radioimmunoprecipitation assays, surface plasmon resonance, and biolayer interferometry, as well as cell-based luciferase reporter gene neutralizing antibody assays.

[0098] In some aspects, the fusion protein or chimeric protein significantly increases the levels, cell counts, and / or amounts of neutrophils, monocytes, eosinophils, total white blood cells, and platelets. In some embodiments, the fusion protein or chimeric protein significantly increases the levels, cell counts, and / or amounts of platelets.

[0099] In some embodiments, the cell recombinant fusion protein or chimeric protein is soluble.

[0100] Nucleic Acids and Host Cells In some embodiments, a nucleic acid molecule is provided that encodes a fusion protein or chimeric protein described herein. In some embodiments, the nucleic acid molecule has a codon optimized sequence.

[0101] In some embodiments, the host cell is a yeast, mammalian, bacterial, insect, algae, or plant cell.

[0102] In some embodiments, the yeast cell is a non-methylotrophic yeast cell. In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

[0103] In some embodiments, human host cells are provided that express the nucleic acid molecules described herein.

[0104] In some embodiments, non-human host cells are provided that express the nucleic acid molecules described herein.

[0105] In some embodiments, a CHO cell is provided that expresses a nucleic acid molecule described herein.

[0106] Pharmaceutical Compositions and Formulations In some embodiments, a pharmaceutical composition is provided comprising a fusion protein or chimeric protein described herein and a pharma- ceutically acceptable excipient or carrier.

[0107] Any pharmaceutical composition described herein can be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier or vehicle. Such compositions can optionally include a suitable amount of a pharma- ceutically acceptable excipient to provide the form for proper administration.

[0108] In various embodiments, the pharmaceutical excipients may be liquids, such as water and oils, e.g., of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like). Pharmaceutical excipients may be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary, stabilizing, thickening, lubricating, and coloring agents may be used. In one embodiment, the pharma-ceutically acceptable excipients are sterile when administered to a subject. When any of the agents described herein are administered intravenously, water is a useful excipient. Saline, aqueous dextrose, and glycerol solutions may also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. The agents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if necessary. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0109] The present disclosure includes the described pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any of the inventive pharmaceutical compositions (and / or additional therapeutic agents) described herein may be in the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquid, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, freeze suspensions, dry powders, or any 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 a further 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.

[0110] If necessary, the pharmaceutical composition of the present invention (and / or the additional therapeutic agent) may also include a solubilizing agent. The agent may also be delivered in a suitable vehicle or delivery device known in the art. The combination therapies outlined herein may be delivered together in a single delivery vehicle or delivery device.

[0111] The formulations containing the pharmaceutical composition of the present invention (and / or additional therapeutic agent) may conveniently be provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods generally include the step of combining the therapeutic agent with the carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately combining the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired dosage form of the formulation (e.g., wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0112] In various embodiments, any pharmaceutical composition described herein (and / or additional therapeutic agent) is formulated in accordance with routine procedures into a composition suitable for the administration methods described herein.

[0113] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, administration is oral. In other embodiments, administration is by parenteral injection. The method of administration can be at the discretion of the physician and depends in part on the site of the medical condition. In most cases, administration will result in release of any agent described herein into the bloodstream.

[0114] In certain embodiments, the fusion protein or chimeric protein or pharmaceutical composition thereof (and / or additional therapeutic agents) is administered via the intravenous route.

[0115] In one embodiment, the pharmaceutical compositions (and / or additional therapeutic agents) described herein are formulated according to routine procedures as compositions suitable for oral administration. Compositions for oral administration may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as, for example, sweeteners, such as fructose, aspartame, or saccharin; flavorings, such as peppermint, wintergreen oil, or cherry; coloring agents; and preservatives, to provide a medicamentically palatable formulation. Additionally, when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the digestive tract, providing a sustained action over a longer period of time. Selectively permeable membranes covering any of the osmotically active driving pharmaceutical compositions (and / or additional therapeutic agents) described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which expands and displaces the drug or drug composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked profiles of immediate release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. In one embodiment, the excipients are pharmaceutical grade. Suspensions can include suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar agar, tragacanth, and the like, and mixtures thereof, in addition to the active compound.

[0116] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. These may contain, for example, suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as EDTA; buffers such as acetate, citrate, or phosphate; tonicity adjusters such as sodium chloride or dextrose, and the like.

[0117] For intravenous administration, suitable carriers include saline, sterile water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0118] The pharmaceutical compositions (and / or additional therapeutic agents) of any of the inventions described herein may be administered by controlled or sustained release means or by delivery devices known to those of skill in the art. Non-limiting examples include those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms are useful for providing controlled or sustained release of one or more active ingredients, for example, using hydropropyl cellulose, hydropropyl methyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof to provide desired release profiles at various ratios. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients of the pharmaceuticals described herein. Thus, the present disclosure provides single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel capsules, and caplets, adapted for controlled or sustained release.

[0119] Controlled or sustained release of an active ingredient can be stimulated by various conditions, including, but not limited to, changes in pH, changes in temperature, stimulation with light of appropriate wavelengths, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0120] In another embodiment, a controlled release system may be placed near the target area to be treated, requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may also be used.

[0121] The pharmaceutical preparations are preferably sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. If the composition is lyophilized, sterile filtration can be performed prior to or following lyophilization and reconstitution.

[0122] Pharmaceutically acceptable salts and excipients The compositions described herein may have a sufficiently basic functional group that can react with inorganic or organic acids to form pharma- ceutically acceptable salts, or a carboxyl group that can react with inorganic or organic bases to form pharma-ceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharma-ceutically acceptable acids as is well known in the art. Such salts include, for example, the pharma-ceutically acceptable salts described in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0123] Pharmaceutically acceptable salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, superphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, benzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate salts.

[0124] The term "pharmaceutically acceptable salt" also refers to a salt of a composition of the present disclosure having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, lithium, etc.; hydroxides of alkaline earth metals such as calcium, magnesium, etc.; hydroxides of other metals such as aluminum, zinc, etc.; ammonia, and unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, organic amines such as dicyclohexylamine; 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-di-lower alkyl-N-(hydroxyl-lower alkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0125] In some embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0126] How to use In one aspect, methods are provided for treating a patient or subject undergoing or having undergone cancer treatment, or a patient or subject undergoing or having undergone a bone marrow transplant, and / or a patient or subject acutely exposed to myelosuppressive amounts of radiation (e.g., Hematopoietic Acute Radiation Syndrome (H-ARS)), with or without other injuries such as burns, wounds, infections, hemorrhage, or blunt trauma (e.g., Radiation Combined Injury (RCI)), comprising administering to the patient a therapeutically effective amount of a fusion protein or chimeric protein of the invention or a pharmaceutical composition thereof. In some embodiments, the patient is treated by modulating the clonal proliferation, survival, differentiation, and activation state of hematopoietic progenitor cells. In some embodiments, the patient is treated by modulating the myelomonocytic cell lineage by promoting the proliferation of megakaryocyte and erythroid progenitor cells. In some embodiments, the patient is treated by modulating the hematopoietic progenitor cells by stimulating the survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells. In some embodiments, patients are treated following bone marrow transplantation by modulating hematopoietic progenitor cells by stimulating survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells.

[0127] In one aspect, a method of treatment is provided comprising administering to a patient a therapeutically effective amount of a fusion or chimeric protein of the invention or a pharmaceutical composition thereof, or contacting cells with an effective amount of a pharmaceutical composition as described herein and administering a therapeutically effective amount of said cells, said treatment promoting neutrophil recovery and / or reducing the incidence of infections after induction chemotherapy; mobilizing hematopoietic progenitor cells to peripheral blood for harvesting by leukapheresis and transplantation; promoting bone marrow reconstitution after autologous or allogeneic bone marrow progenitor or peripheral blood progenitor cell transplantation; treating delayed neutrophil recovery or engraftment failure after autologous or allogeneic bone marrow transplantation; and / or treating hematopoietic acute radiation syndrome (H-ARS); and / or treating combined radiation injury (RCI).

[0128] In one aspect, a method for treating a viral infection is provided, comprising administering to a patient in need thereof an effective amount of a composition comprising the fusion protein or chimeric protein of the invention or a pharmaceutical composition comprising same.

[0129] In some embodiments, the viral infection is an influenza infection, optionally selected from influenza A, B, C, and D virus infections.

[0130] In some embodiments, the viral infection is a viral hemorrhagic fever, optionally caused by a group of viruses selected from arenaviruses, flaviviruses, filoviruses, hantaviruses, nairoviruses, and fenuviruses.

[0131] In some embodiments, the pulmonary infection is pneumonic plague caused by infection with Yersinia pestis.

[0132] In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the coronavirus is a betacoronavirus, 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. In some embodiments, the coronavirus is an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

[0133] Coronaviruses are members of the Coronaviridae family, which includes the Betacoronaviruses and Alphacoronaviruses respiratory pathogens that have become known to invade humans relatively recently. The Coronaviridae family includes Betacoronaviruses, 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. Alphacoronaviruses include, for example, HCoV-NL63 and HCoV-229E.

[0134] Coronaviruses enter cells via a "spike" surface glycoprotein responsible for viral recognition by angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor in mammalian hosts that facilitates viral entry into host cells. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020. Novel coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2.

[0135] SARS-CoV-2 is a new virus thought to have originated from bats. COVID-19 causes severe respiratory distress and this RNA virus strain is responsible for the recent pandemic, which has been declared a major threat to public health and a global emergency. Phylogenetic analysis of the entire genome of SARS-CoV-2 revealed that the virus is most closely related (89.1% nucleotide similarity) to the group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020.

[0136] SARS-CoV-2 is an enveloped, single-stranded RNA virus that encodes the "spike" protein, also known as the S protein, a surface glycoprotein that mediates binding to cell surface receptors, transmembrane proteins, envelope proteins, and nucleocapsid proteins. The S protein, which includes the S1 and S2 subunits, is a trimeric class I fusion protein that exists in a prefusion conformation that undergoes structural rearrangement to fuse the viral membrane with the host cell membrane. See, e.g., Li, F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 3: 237-261(2016), which is incorporated herein by reference in its entirety. The structure of the SARS-CoV-2 spike protein in the prefusion conformation has been discovered. See, e.g., 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.

[0137] Phylogenetic analysis of the complete genome of SARS-CoV-2 (GenBank Accession No.: MN908947) revealed that the virus is most closely related (89.1% nucleotide similarity) to the group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020, incorporated herein by reference in its entirety.

[0138] SARS-CoV-2 has spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphoprotein N. The complete genome of SARS-CoV-2 coronavirus (29903 nucleotides, single-stranded RNA) is listed in the NCBI database as GenBank Reference Sequence: MN908947. The coronavirus protein can be selected from 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 nucleocapsid phosphoprotein E (GenBank Reference Sequence: QHD43423).

[0139] In some embodiments, the method prevents or reduces the development of acute respiratory distress syndrome (ARDS) in a patient.

[0140] In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the patient is suffering from COVID-19. In some embodiments, the patient is suffering from one or more of fever, cough, shortness of breath, diarrhea, upper respiratory tract symptoms, lower respiratory tract symptoms, pneumonia, and acute respiratory syndrome.

[0141] In some embodiments, the patient is hypoxic. In some embodiments, the patient is suffering from respiratory distress. In some embodiments, the method improves oxygenation of the patient. In some embodiments, the method prevents or mitigates the transition of the patient from respiratory distress to cytokine imbalance. In some embodiments, the method reverses or prevents a cytokine storm. In some embodiments, the method reverses or prevents a pulmonary or systemic cytokine storm. In some embodiments, the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis. In some embodiments, the method reverses or prevents excessive production of one or more inflammatory cytokines. In some embodiments, the inflammatory cytokine is one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

[0142] In some embodiments, the methods cause a decrease in the patient's viral or bacterial load compared to before treatment.

[0143] In some embodiments, the viral infection is selected from a betacoronavirus infection, optionally selected from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a severe acute respiratory syndrome coronavirus (SARS-CoV-1), a Middle East respiratory syndrome coronavirus (MERS-CoV), an HCoV-HKU1, and an HCoV-OC43 infection. In some embodiments, the viral infection is selected from an alphacoronavirus infection, optionally selected from an HCoV-NL63 and an HCoV-229E infection.

[0144] In some embodiments, the betacoronavirus infection is severe acute respiratory syndrome (SARS).

[0145] In some embodiments, the betacoronavirus infection is or is associated with coronavirus disease 2019 (COVID-19).

[0146] In some embodiments, the viral infection is an influenza infection, optionally selected from influenza virus infections A, B, C, and D. In some embodiments, the influenza infection is pandemic 2009 influenza A (H1N1) or avian influenza A (H5N1).

[0147] In some embodiments, the viral infection is a viral hemorrhagic fever, optionally caused by a group of viruses selected from arenaviruses, flaviviruses, filoviruses, hantaviruses, nairoviruses, and fenuviruses.

[0148] In some embodiments, the pulmonary infection is pneumonic plague caused by infection with Yersinia pestis.

[0149] In one aspect, a method for treating symptoms caused by exposure to a chemical weapon is provided, comprising administering to a patient in need thereof an effective amount of a composition comprising the fusion protein or chimeric protein of the invention or a pharmaceutical composition comprising same.

[0150] In some embodiments, the chemical weapon is sulfur mustard gas (SM / HD) and / or chlorine gas.

[0151] In some embodiments, the symptoms of exposure in said patient are characterized by myelosuppression.

[0152] In some embodiments, the methods provided cause a reduction and / or amelioration of symptoms, including bone marrow suppression, caused by exposure to sulfur mustard gas (SM / HD) and / or chlorine gas.

[0153] In one aspect, there is provided a method for treating and / or ameliorating symptoms caused by a neurodegenerative disease in a patient or subject, comprising administering to the patient a therapeutically effective amount of a fusion protein or chimeric protein of the invention or a pharmaceutical composition thereof.

[0154] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), dementia with Lewy bodies, Parkinson's disease dementia, epilepsy, stroke, Huntington's disease, cerebral hypoxia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and peripheral neuropathy.

[0155] In some embodiments, the patient's neurodegenerative disease is characterized by oxidative stress, loss of neurite integrity, apoptosis, neuronal loss, or / and an inflammatory response, hi some embodiments, the neurodegenerative disease is associated with cognitive impairment.

[0156] In one aspect, a method of treatment is provided comprising administering to a patient suffering from a neurodegenerative disease a therapeutically effective amount of a fusion or chimeric protein of the invention or a pharmaceutical composition thereof, or contacting cells with an effective amount of a pharmaceutical composition as described herein and administering a therapeutically effective amount of said cells, wherein said treatment induces a disease-modifying response; slows, temporarily or permanently, the decline in cognitive function; causes an improvement in symptoms; and / or delays the onset and / or development of the disease.

[0157] In one aspect, the fusion protein or chimeric protein of the invention is provided as a composition that promotes enhanced delivery of recombinant GM-CSF through endothelial capillaries (blood-brain barrier, BBB) for central nervous system (CNS) treatment.

[0158] In one aspect, there is provided a method for treating and / or ameliorating symptoms caused by an autoimmune disease in a patient or subject, comprising administering to the patient a therapeutically effective amount of a fusion protein or chimeric protein of the invention or a pharmaceutical composition thereof.

[0159] In one embodiment, the autoimmune disease is optionally selected from Crohn's disease and / or ulcerative colitis (UC) and / or irritable bowel disease (IBD).

[0160] In one aspect, a method of treatment is provided comprising administering to a patient suffering from an autoimmune disease a therapeutically effective amount of a fusion protein or chimeric protein of the present invention or a pharmaceutical composition thereof, or contacting a cell with an effective amount of a pharmaceutical composition as described herein and administering a therapeutically effective amount of said cell, said method of treatment may restore the balance between effector and regulatory immune function.

[0161] In one aspect, a method of treating and / or ameliorating symptoms in a patient or subject caused by a caspase recruitment domain family member 9 (CARD9) deficiency is provided.

[0162] In one aspect, there is provided a method of treating a patient or subject undergoing or having undergone treatment for a fungal infection, comprising administering to the patient a therapeutically effective amount of a fusion protein or chimeric protein of the invention or a pharmaceutical composition thereof.

[0163] In some embodiments, the compositions of the invention are used as an adjunct to antifungal therapy, hi some embodiments, the compositions of the invention decrease and / or reduce fungal burden in various tissues.

[0164] In one aspect, a method of treating a patient or subject undergoing or having been treated for hematopoietic syndrome of acute radiation syndrome (H-ARS) is provided.

[0165] In one aspect, a method of treating a patient or subject undergoing or having undergone treatment for combined radiation injury (RCI) is provided.

[0166] In one aspect, a method of treating a patient or subject undergoing or having undergone nerve transection is provided.

[0167] In one aspect, a method of treating a patient or subject undergoing or having undergone treatment for a traumatic brain injury is provided.

[0168] In some embodiments, the compositions of the invention are used as an adjunct to antifungal therapy, hi some embodiments, the compositions of the invention decrease and / or reduce fungal burden in various tissues.

[0169] In one aspect, methods are provided for treating a patient or subject undergoing or having undergone treatment for sepsis. In some embodiments, the compositions of the invention balance and / or modulate inflammation and the acute phase response, reducing morbidity and mortality.

[0170] In one aspect, a method of treating a patient or subject undergoing or having been treated for multiple organ dysfunction syndrome (MODS) is provided.

[0171] In one aspect, a method of treating a patient or subject who is undergoing or has undergone treatment for peripheral artery disease / peripheral arterial disease is provided.

[0172] In one aspect, a method of treating a patient or subject undergoing or having undergone treatment for chronic wound healing is provided.

[0173] In one embodiment, said chronic wound is selected from non-healing wounds, optionally due to leprosy, leg ulcers, and / or other various causes.

[0174] In some embodiments, the compositions of the present invention are used as adjunct therapy to wound healing therapies, including but not limited to hyperbaric oxygen therapy, ultrasound and electromagnetic therapy, and / or negative pressure wound therapy. In some embodiments, the compositions of the present invention act on various epidermal cells to promote wound healing. In some embodiments, the compositions of the present invention promote tissue remodeling and regeneration.

[0175] Combination Therapies and Additional Therapeutic Agents In various embodiments, the pharmaceutical compositions of the present disclosure are co-administered in combination with additional agents. The co-administration may be simultaneous or sequential.

[0176] In one embodiment, the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof of the present disclosure are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof can be performed by simultaneous administration of a single formulation (e.g., a formulation comprising the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof) or by simultaneous administration of separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the fusion protein or chimeric protein).

[0177] In the case of combined administration, the therapeutic agents do not need to be administered simultaneously, so long as the pharmacological activities of the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the targeting moiety, fusion protein or chimeric protein or pharmaceutical composition thereof, composition may be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the fusion protein or chimeric protein are administered at a time interval of about 60 minutes or more. For example, the time between the sequential administration of the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof may be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, more than about 2 weeks, or more than about 1 month. The optimal administration time depends on the rate of metabolism, excretion, and / or pharmacodynamic activity of the additional therapeutic agent and the fusion protein or chimeric protein or pharmaceutical composition thereof to be administered. Either the additional therapeutic agent and the fusion protein or chimeric protein composition or pharmaceutical composition thereof may be administered first.

[0178] Concomitant administration of the therapeutic agents does not require that the therapeutic agents be administered to a subject by the same route of administration, rather, each therapeutic agent may be administered by any suitable route, such as parenterally or parenterally.

[0179] In some embodiments, the fusion or chimeric proteins or pharmaceutical compositions thereof described herein act synergistically when combined with other therapeutic agents, In such embodiments, the targeting moiety, the composition of the invention, and the additional therapeutic agent may be administered at lower doses than would be used if those agents were used in a monotherapy context.

[0180] In some embodiments, the additional therapeutic agent is an antiviral agent.

[0181] In some embodiments, the additional therapeutic agent is remdesivir, favipiravir, oseltamivir, baloxavir, galidesivir, amprenavir, tipranavir, saquinavir, nelfinavir, indinavir, darunavir, atazanavir, emetine, lopinavir, and / or ritonavir, arbidol, and lopinavir / ritonavir, and / or ribavirin, darunavir, and cobicistat, and / or or IFN-β-1b, β-D-N4-hydroxycytidine (NHC), antiviral agents such as EIDD-1931 or EIDD-2801 or EIDD-2801; glucocorticoids, IFN-α2a, IFN-α2b, IFN-β, PEGylated IFN-γ, baricitinib, sirolimus, clazakizumab, canakinumab, XPro1595, tocilizumab, sarilumab, siltuximab, a immunomodulators such as darimumab, eculizumab, ivermectin, anakinra, plescovix, Xiyanping, fingolimod, methylprednisolone, leronlimab, thalidomide, MK-2206, nicolasamide, nitazoxamide, chloroquine, or hydroxychloroquine; antibiotics such as kalimycin, brilacidin, azithromycin, or valinomycin; angiotensin inhibitors / antagonists such as rhACE2 / GSK2586881 / APN01, losartan, eprosartan, telmisartan, or valsartan; serine protease inhibitors such as camostat mesylate or nafamostat; or other agents such as bromhexine, aprotinin, chlorpromazine, zotatifin, methotrexate, lenalidomide, anti-VEGF-A, and intravenous immunoglobulin (IVIG). For example, in some embodiments, any of these additional therapeutic agents are used in the context of SARS-CoV-2 infection.

[0182] In some embodiments, the additional therapeutic agent is selected from favipiravir, laninamivir octanoate, peramivir, zanamivir, oseltamivir phosphate, baloxavir marboxil, umifenovir, amantadine urmine hydrochloride, rimantadine hydrochloride, adapromine, LASAG / BAY81-87981, celecoxib, etanercept, metformin, gemcitabine, dapivirine, trametinib, lisinopril, naproxen, nalidixic acid, dorzolamide, ruxolitinib, midodrine, diltiazem; statins such as atorvastatin, nitazoxanide; PPAR antagonists such as gemfibrozil. For example, in some embodiments, any of these additional therapeutic agents are used in the context of influenza infections or other viral infections such as those causing viral hemorrhagic fevers.

[0183] In some embodiments, the additional therapeutic agent is an agent for treating a neurodegenerative disease.

[0184] In some embodiments, the additional therapeutic agent is selected from levodopa; cholinesterase inhibitors including donepezil (ARICEPT), rivastigmine (EXELON), galantamine (RAZADYNE); atypical / second generation antipsychotics such as serotonin-dopamine antagonists (SDAs), multi-receptor targeted antipsychotics (MARTAs), and D2 partial agonists (e.g., ABILIFY / aripiprazole); NMDA receptor antagonists memantine, riluzole (RILUTEK), NSAIDs (nonsteroidal anti-inflammatory drugs), caffeine A2A receptor antagonists, and CERE-120 (adeno-associated virus serotype 2-neurturin), and deep brain stimulation. For example, in some embodiments, any of these therapeutic agents are used in the context of a neurodegenerative disease.

[0185] In some embodiments, the additional therapeutic agent is an antifungal agent.

[0186] In some embodiments, the additional therapeutic agent is selected from nystatin, amphotericin B, terbinafine, almamentarium, a pyrimidine analogue such as flucytosine, a triazole such as fluconazole, isavuconazole, itraconazole, pramiconazole, ravuconazole, voriconazole, and posaconazole, an imidazole such as clotrimazole, econazole, ketoconazole, and miconazole, and an echinocandin such as caspofungin, micafungin, and anidulafungin. For example, in some embodiments, any of these therapeutic agents are used in the context of a fungal infection.

[0187] In some embodiments, the additional therapeutic agent is an agent for treating an autoimmune disease.

[0188] In some embodiments, the additional therapeutic agent is a corticosteroid such as prednisone and budesonide (ENTOCORT EC); an oral 5-aminosalicylates such as sulfasalazine (AZULFIDINE); an anti-inflammatory / anti-inflammatory / immunosuppressant such as azathioprine (AZASAN, IMURAN), mercaptopurine (PURINETHOL, PURIXAN), methotrexate (TREXALL), a corticosteroid; and mesalamine (ASACOL HD). Aminosalicylates such as methotrexate (DELZICOL), balsalazide (COLAZAL), tofacitinib (XELJANZ), cyclosporine (GENGRAF, NEORAL, SANDIMMUNE), and olsalazine (DIPENTUM); The therapeutic agent is selected from biologics such as tetanus io), infliximab (REMICADE), adalimumab (HUMIRA), certolizumab pegol (CIMZIA), ustekinumab (STELARA), and golimumab (SIMPONI); antibiotics such as ciprofloxacin (CIPRO) and metronidazole (FLAGYL); antidiarrheals such as loperamide (IMODIUM AD); pain relievers; vitamins, and supplements such as iron supplements. For example, in some embodiments, any of these therapeutic agents are used in the context of an autoimmune disease.

[0189] In some embodiments, the additional therapeutic agent is an agent that promotes or assists in wound healing or other skin conditions.

[0190] In some embodiments, the additional therapeutic agent is selected from the group consisting of hyperbaric oxygen therapy, ultrasound and electromagnetic therapy, and / or negative pressure wound therapy; corticosteroids such as hydrocortisone, triamcinolone (ACETONIDE, TRIANEX), clobetasol (TEMOVATE); vitamin D analogs such as calcipotriene and calcitriol (VECTICAL); retinoids such as tazarotene (TAZORAC, AVAGE) and acitretin (SORIATANE); calcineurin inhibitors such as tacrolimus (PROTOPIC) and pimecrolimus (ELIDEL); salicylic acid; coal tar; Geckermann therapy; anthralin; natural light, UVB broadband, UVB narrowband, psoralen + ultraviolet A phototherapy such as PUVA, and excimer laser therapy; immunosuppressants such as steroids, methotrexate (TREXALL), adalimumab (HUMIRA), infliximab (REMICADE), and cyclosporine; biologics such as etanercept (ENBREL), infliximab (REMICADE), adalimumab (HUMIRA), ustekinumab (STELARA), secukinumab (COSENTYX), and ixekizumab (TALTZ); thioguanine (TABLOID), and hydroxyurea (DROXIA, HYDREA), aloe extract cream, fish oil supplements, Oregon grape, and essential oils.

[0191] In some embodiments, the additional therapeutic agent is a cardiovascular disease or peripheral arterial disease medication.

[0192] In some embodiments, the additional therapeutic agent is an anticoagulant, such as apixaban (ELIQUIS), warfarin (COUMADIN); an antiplatelet agent, such as aspirin and a P2Y12 inhibitor (clopidogrel (PLAVIX), prasugrel (EFFIENT), ticagrelor (BRILINTA), dipyridamole (PERSANTINE), etc.), and dual antiplatelet therapy (DAPT). angiotensin-converting enzyme (ACE) inhibitors such as benazepril (LOTENSIN); angiotensin II receptor blockers (or inhibitors) such as azilsartan (EBARBI); angiotensin receptor neprilysin inhibitors (ARNI) such as sacubitril / valsartan (ENTRESTO); beta-blockers (beta-adrenergic blockers) such as acebutolol (SECTRAL); carvedilol (COREG, COREG combination of alpha-blockers and beta-blockers such as amlodipine (Norvasc); cholesterol-lowering drugs such as statins (atorvastatin / Lipitor) and nicotinic acid (niacin); cholesterol absorption inhibitors (ezetimibe / Zetia); combination of statins and cholesterol absorption inhibitors (ezetimibe / simvastatin (Vytorin); digitalis (digoxin / Lanoxin); diuretics such as acetazolamide (Diamox); and vasodilators such as isosorbide dinitrate (ISORDIL) and nitroglycerin (NITRO BID, NITRO STAT).

[0193] In some embodiments, the additional therapeutic agent is an agent for treating sepsis or septicemia.

[0194] In some embodiments, the additional therapeutic agent is selected from antibiotics such as ceftriaxone (ROCEPHIN), cefotaxime (CLAFORAN), ampicillin, and sulbactam (UNASYN), ceftazidime (FORTAZ), and clindamycin (CLEOCIN).

[0195] In some embodiments, the additional therapeutic agent is an agent for treating an oncological indication.

[0196] In some embodiments, the additional therapeutic agent is selected from cytotoxic agents, including toxins such as ricin; chemotherapeutic agents, such as 5-fluorouracil; radioisotopes (e.g., iodine-131, radium-223); targeted therapeutic agents, such as HERCEPTIN; and immuno-oncology agents, such as ipilimumab (YERVOY) and nivolumab (OPDIVO).

[0197] In some embodiments, the additional therapy is an agent to increase the number of platelet cells or platelet count and / or treat immune thrombocytopenia (ITP).

[0198] In some embodiments, the additional therapeutic agent is selected from steroids such as prednisone, immunoglobulin therapy, romiplostim (NPLATE), eltrombopag (PROMACTA), and rituximab (RITUXAN, TRUXIMA), and the like.

[0199] Method of preparation In some embodiments, methods are provided for making a fusion or chimeric protein, comprising: (a) obtaining cells or extracts thereof transfected with a nucleic acid encoding recombinant human GM-CSF comprising an amino acid sequence having at least about 97% identity to SEQ ID NO:1 or SEQ ID NO:2, optionally with a substitution or deletion at position N37 or a position corresponding thereto, a linker, and a humanized single domain antibody; (b) purifying the fusion or chimeric protein from the transfected cells using one or more HPLC columns; and (c) collecting the purified fusion or chimeric protein, wherein the purified chimeric protein is substantially free of hypermannosylated GM-CSF forms.

[0200] In some embodiments, the cell is a prokaryotic or eukaryotic host cell, such as a yeast cell, a mammalian cell, a bacterial cell, an insect cell, an algae cell, or a plant cell.

[0201] Suitable prokaryotic host cells include bacterial cells (e.g., E. coli, Bacillus subtilis, Mycobacterium spp., M. tuberculosis, or other suitable bacterial cells), and archaeal cells (e.g., Methanococcus jannaschii and Methanococcus maripaludis).

[0202] In some embodiments, the host cell of the present disclosure is a eukaryotic host cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, animal cells (e.g., mammalian cells, avian cells, fish cells), and plant cells.

[0203] Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, and the like.

[0204] Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti, and the like.

[0205] Suitable yeast host cells include, but are not limited to, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0206] Suitable filamentous fungal host cells include, for example, any filamentous form of the subdivision Eumycotina and Oomycota. In some embodiments, the filamentous fungal host cell is selected from the group consisting of Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella, or teleomorphs, anamorphs, synonyms, or taxonomic equivalents thereof. In one embodiment, the filamentous fungus is selected from Aspergillus of the A. nidulans, A. oryzae, A. sojae, and A. niger groups. In one embodiment, the filamentous fungus is Aspergillus niger.

[0207] In some embodiments, methods are provided for producing a composition comprising a fusion protein or chimeric protein, the method comprising: (a) obtaining a cell or an extract thereof transfected with a nucleic acid encoding a recombinant human GM-CSF comprising an amino acid sequence having at least about 97% identity to SEQ ID NO:2 and having a substitution or deletion at position N37 or a position corresponding thereto, a linker, and a VHH humanized Nanobody; (b) purifying the fusion protein or chimeric protein from the transfected cell using one or more HPLC columns; and (c) recovering the purified fusion protein or chimeric protein, wherein the purified fusion protein or chimeric protein is substantially free of hypermannosylated GM-CSF forms.

[0208] In some embodiments, the cell is a yeast cell, such as, but not limited to, Saccharomyces cerevisiae.

[0209] In some embodiments, the cell is a mammalian cell, such as, but not limited to, a Chinese Hamster Ovary (CHO) cell.

[0210] In some embodiments, production of the fusion or chimeric protein in mammalian cells increases expression levels of the composition as compared to production using non-mammalian cells.

[0211] In some embodiments, the method further comprises formulating the purified fusion protein or chimeric protein for injection, for example, subcutaneous or intravenous injection.

[0212] array SEQ ID NO: 1 is wild-type GM-CSF. Substitution or deletion sites in some embodiments are underlined and in bold.

[0213] [ka]

[0214] SEQ ID NO:2 is sargramostim. Substitution or deletion sites in some embodiments are underlined and in bold.

[0215] [ka]

[0216] SEQ ID NO:3 is the human VH3-23 sequence.

[0217] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

[0218] SEQ ID NO:4 is the G4S linker sequence.

[0219] [ka]

[0220] SEQ ID NO:5 is the G4A linker sequence.

[0221] [ka]

[0222] SEQ ID NO:6 is a variant of the hinge sequence of the human IgG1 sequence.

[0223] EPKSSDKTHTSPPSPAPE

[0224] SEQ ID NO: 7 is the VHH-GMCSF construct sequence, with an anti-HSA VHH at the N-terminus, GM-CSF at the C-terminus, and G4S as a linker.

[0225] [ka]

[0226] SEQ ID NO:8 is the VHH-GMCSF construct sequence, with an anti-HSA VHH at the N-terminus, GM-CSF at the C-terminus, and a hinge as a linker.

[0227] [ka]

[0228] SEQ ID NO: 9 is the VHH-GMCSF construct sequence, with GM-CSF at the N-terminus, an anti-HSA VHH at the C-terminus, and a hinge as a linker.

[0229] [ka]

[0230] SEQ ID NO: 10 is the VHH-GMCSF construct sequence, with GM-CSF at the N-terminus, an anti-HSA VHH at the C-terminus, and G4S as a linker.

[0231] [ka]

[0232] SEQ ID NO: 11 is the VHH-GMCSF construct sequence, with an anti-HSA VHH at the N-terminus, GM-CSF at the C-terminus, and G4A as a linker.

[0233] [ka]

[0234] SEQ ID NOs:12 to 14 are the CDRs: CDR-H1: GETLDYYA (SEQ ID NO:12), CDR-H2: IASSGGST (SEQ ID NO:13), and CDR-H3: AAAVLECRTVVRGYDY (SEQ ID NO:14).

[0235] SEQ ID NOs: 15 to 17 are CDRs: CDR-H1: ETLDYYAIG (SEQ ID NO: 15), CDR-H2: IASSGSTNYADSVKG (SEQ ID NO: 16), CDR-H3: AVLECRTVVRGYDY (SEQ ID NO: 17).

[0236] definition In connection with the invention disclosed herein, the following definitions are used: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0237] An "effective amount" as used in reference to an agent effective to treat a coronavirus infection is an amount effective to treat or alleviate a coronavirus infection.

[0238] As used herein, "a," "an," or "the" can mean one or more than one. Also, the term "about" when used in connection with a referenced numerical indication means the referenced numerical indication plus or minus up to 10% of the referenced numerical indication. For example, the term "about 50" covers the range of 45 to 55.

[0239] As referred to herein, all composition percentages are by weight of the total composition unless otherwise specified. As used herein, the word "include" and variations thereof are non-limiting, and the description of an item in a list does not exclude other similar items that may be useful in the materials, compositions, devices, and methods of the present technology. Similarly, the words "can" and "may" and variations thereof are non-limiting, and a description that an embodiment may or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0240] Although the open-ended term "comprising" is used herein as a synonym for terms such as "including," "containing," "having," and the like, to describe and claim the invention, the invention or embodiments thereof may instead be described using alternative terms such as "consisting of" or "consisting essentially of."

[0241] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0242] Example 1: Discovery and humanization of VHH molecules Two llamas were immunized with human serum albumin (HSA) to generate antibodies. Total RNA was isolated from the cells and cDNA was subsequently synthesized. This cDNA was used to amplify the variable domain of the heavy chain of the camelid heavy chain antibody (VHH) coding sequence by PCR. The structure of heavy chain IgG (hcIgG) is illustrated in Figure 1A.

[0243] Subsequently, fluorescence-activated cell sorting (FACs) was used in combination with fluorescently labeled human serum albumin (HSA) binding to yeast expressing VHHs that bind HSA to isolate individual VHHs that bind HSA. The yeast surface display system for screening of antigen-binding scaffold libraries is shown in Figure 1B. These VHHs did not bind albumin with epitopes that block FcRn binding, important for albumin recycling and pharmacokinetic (pK) properties. The clones were also shown not to bind nonspecifically to membrane preparations. Finally, the binding properties of the VHH clones did not change when measured between pH 5.5 and 7.4. After identification of HSA-binding VHH clones, the VHHs were humanized by appropriate backmutations and CDR grafting onto the appropriate human VHHs, together with the llama-specific sequences necessary to maintain the stability of the VHHs.

[0244] To demonstrate that VHHs promote serum half-life extension, the three identified and humanized anti-HSA VHH clones were used in experimental fusion proteins. Protein fusion molecules were generated to investigate the potential half-life extension capabilities of the three VHHs. Fab molecule fusion proteins were constructed with each of the three VHHs. VHH-promoted half-life extension of Fab fragments was demonstrated in a mouse model, demonstrating a 17-fold increase in Fab half-life. VHH59-1 also extends the half-life of other anti-HSA-VHH molecules by more than two-fold (Figure 1C).

[0245] Example 2: Identification and construction of GMCSF-VHHs Four variants were initially generated and characterized. The orientation of VHH and GMCSF was varied around two linker sequences: VHH-linker-GMCSF or GMCSF-linker-VHH. (GGGGS)x4 or (GGGGA)x4 linkers were used. The second sequence is a modified hinge sequence of human IgG1, since in llamas the VHH is directly linked to the hinge region of IgG (i.e. the junction of the VHH and constant region of the llama's native antibody). The albumin leader sequence was chosen instead of the native GMCSF leader, since the data showed that the leader cleavage was heterogeneous, whereas the albumin leader sequence was cleaved uniformly, as assessed using SignalP software analysis. The four constructs were cloned and transiently expressed in CHO cells. The secretory leader sequence was the same for all four constructs. The sequences are shown in Figure 2.

[0246] Example 3: VHH-GA-GMCSF * Molecular characterization Multiple variations of anti-HSA-VHH59-1 were generated to empirically determine which variants had the best exploitable properties, including expression titers, aggregation as determined by size exclusion chromatography, ability of the VHH to bind to HSA in the context of a fusion or chimeric protein, biological activity of the fused GMCSF, and binding properties to the cognate receptor of GMCSF. *The GM-CSF sequence was identical to the GMCSF variant sequence previously used to express a CHO-derived GM-CSF variant.

[0247] The expression titer levels in transient CHO of the four constructs are shown in Table 1. The expression level of N-terminal GM-CSF was much higher than that of C-terminal. A 30% reduction in yield was observed due to the relatively weak binding between VHH and ProA resin. However, this GMCSF-hinge-VHH also appeared to contain more aggregates compared to the other constructs. [Table 1]

[0248] The ability of the VHH moieties of the fusion or chimeric proteins to bind to human serum albumin (HSA) using a ForteBio-based assay was evaluated. Interestingly, all four VHH-GMCSF fusion or chimeric proteins were only able to bind to HSA when immobilized on the sensor and albumin was in solution, but not when albumin was mobile on the sensor.

[0249] The ability of GM-CSF in the fusion or chimera to target its cognate receptor was assessed in a bioactivity assay using TF-1 cells in the presence (Figure 3A) and absence (Figure 3B) of albumin. TF-1 cells proliferated in response to exposure to fusion or chimeric GM-CSF. The four chimeric or fusion proteins were co-immunopreserved with CHO-derived GM-CSF. *The VHH-G4Sx4-GM-CSF variant (VHH-GMCSF) was compared with LEUKINE and commercial GM-CSF-containing constructs. The data showed that VHH-G4Sx4-GM-CSF and GM-CSF-G4Sx4-VHH were biologically active in the TF-1 cell-based assay, i.e., cells proliferated when GM-CSF fusion or chimeric proteins were added to the culture medium. On the other hand, the GMCSF-G4Sx4-VHH EC50 (Table 2) was confirmed to be slightly lower or less active than the reversed one. CHO-derived GM-CSF * Fusion or chimeric proteins containing GM-CSF showed activity similar to LEUKINE. Thus, our results indicate that the orientation of GM-CSF relative to the VHH in the fusion or chimeric proteins affects biological activity in cell-based assays, with greater inhibition of GM-CSF activity in the presence of albumin, both in the presence and absence of albumin. Without intending to be bound by theory, it is possible that binding of albumin to the VHH alters the ability of GM-CSF (at the N-terminus) to interact with its receptor, resulting in a failure to induce proliferation of TF1 cells compared to GM-CSF alone or when GM-CSF was fused to the C-terminus of the VHH linker (Figure 3A-B). [Table 2]

[0250] Example 4: Mass spectrometric evaluation of glycoprofiles of fusion proteins Mass spectrometry was used to verify the heterogeneity of the N-terminal truncation and glycosylation of the molecules. Mass spectrometry confirmed the addition of xylose to the (G4S×4) linker, which is known to be immunogenic in humans. See Arch Biochem Biophys. 426:266-278. Mass spectrometry confirmed that the known glycosylation sites of GM-CSF, Ser7, Ser10, and N27, were also glycosylated in all VHH-G4S×4-GM-CSF variants, indicating that the CHO-produced GM-CSF used in the fusion proteins was glycosylated at the same site. * Six different stable lines were analyzed and the results are summarized in the table below. The intact molecular weight and glycan profile was determined using high-resolution liquid chromatography-mass spectrometry (LC-MS) (Vanquish UPLC Q Exactive, Thermo) (Figure 4A). The figure shows the glycan analysis by mass spectrometry, specifically VHH-G4A GMCSF. * Glycosylation occupancy of six stable CHO clones producing β-lactam β-lactam 1 (C100) is shown. 100 μg of sample was diluted to 2 mg / mL in deionized water and injected into the LC-MS. Data were analyzed and deconvoluted with BioPharmaFinder software to obtain the intact protein molecular weight. Glycosylation species were identified by accurate mass shifts and manual calculations. Based on the intensity of all identified ions, N / O glycosylation occupancy was estimated under the assumption of equal ionization efficiency (Table 3). [Table 3]

[0251] Constructs generated from these six clones were analyzed in a TF-1 cell-based assay. The data showed that activity did not vary when the N-linkage occupancy of N27 varied between approximately 12% and approximately 53%. Thus, the activity of the molecule was not altered or affected by the presence or absence of glycans (Figure 4B and Table 4). [Table 4]

[0252] Example 5: In vitro binding characterization of VHH-GMCSF constructs To assess binding to albumin, VHHs were cloned onto human IgG1 Fc and hinge for ease of purification and analysis. Binding affinities of VHH-Fc constructs were determined by BLI using an Octet 384 (Pall Life Sciences). VH-Fc constructs were loaded onto the AHC sensor and exposed to monomeric targets human, mouse, or rhesus albumin in solution for association and subsequent dissociation in buffer (Table 5 and Figure 5A-C). [Table 5]

[0253] To determine dissociation constants, binding and dissociation reactions were carried out at the indicated pH and data acquisition was carried out at 30°C.

[0254] The graphs show response units (target binding to VHH-Fc on the chip sensor) on the Y-axis and time on the X-axis (Figure 6A-D). The data (time vs. response units) were analyzed using ForteBio data analysis software. The data were fitted to a 1:1 binding model to obtain the binding kinetics (K on ) and dissociation rate (K off ) was calculated and the kd / ka ratio was used to calculate the KD. Table 6 shows the measured and calculated parameters for each target and each pH. [Table 6]

[0255] Example 6: VHH-GA-GMCSF * In vivo characterization of VHH-GA-GMCSF * and two control molecules, CHO-produced GMCSF. *and Leukine were tested in monkey experiments (one monkey per molecule tested). * 20μg for leukin or CHO-produced GM-CSF * After intravenous injection of 10 μg, the pK and biological effects on circulating hematopoietic cells were measured over time. * GM-CSF * Since VHH-GA-GMCSF has a molecular weight approximately twice that of VHH-GA-GMCSF, it was administered at a higher concentration, i.e., the amount was doubled to give an equimolar amount of the molecule. Blood samples were taken 12 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h after injection. The data (Figures 7A-C) show that VHH-GA-GMCSF * The VHH of the construct showed a significant increase in pK from 0.79 h to more than 10 h. Leukine and CHO-produced GMCSF * has a similar half-life (t 1 / 2 ) indicating that CHO production of GMCSF does not affect half-life extension. One rhesus monkey was intravenously injected with 10 μg / Kg of the described form of GMCSF. Blood was collected 12 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, 48 hr, and 72 hr after injection. Blood was analyzed for cellular content and the concentration of the construct in blood was measured by a dedicated construct-specific ELISA. VHH-GA-GMCSF * is approximately 17-fold greater than either leukine or the CHO-produced GMCSF form used in the VHH-GA-GMCSF construct. 1 / 2 Shows. A complete hematopoietic evaluation of cell numbers and cell types after blood collection from the injected monkeys was performed using FACS analysis with cell-specific markers (Figure 8A-E). The number of neutrophils, monocytes, eosinophils, total leukocytes, and platelets were measured (Figure 8A-E). CHO-produced GM-CSF * Both fusion or chimeric VHH-GA-GMCSF and LEUKINE caused similar spikes in neutrophil, monocyte, and white blood cell counts 24 hours after injection, returning to baseline by day 4. *Neutrophil, monocyte, and white blood cell counts peaked on day 2 and returned to baseline between days 8 and 12. The longer persistence of neutrophils, monocytes, and white blood cells after injection of the fusion or chimeric protein indicates that the enhanced pk shown in Figures 7A-C led to a longer duration of biological indicators of increased circulating blood cell counts. Similar delayed kinetics were observed for eosinophilia after injection of the fusion or chimeric protein. The number of neutrophils, monocytes, eosinophils, and white blood cells were also increased after injection of the chimeric or fusion protein. Surprisingly, the data showed that CHO-produced GM-CSF * and LEUKINE, compared to either the fusion or chimeric VHH-GA-GMCSF * A significant increase in platelet count was observed after injection of

[0256] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed by the scope of the following claims.

[0257] Incorporated by reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0258] All headings used herein are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of each section are equally applicable to all sections.

Claims

1. A composition comprising a fusion or chimeric protein comprising a recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) protein, a linker, and a humanized single domain antibody.

2. 2. The composition of claim 1, wherein the recombinant human GM-CSF protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:1 or SEQ ID NO:

2.

3. The composition of claim 2, wherein the recombinant human GM-CSF protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:1 or SEQ ID NO:2, and a substitution or deletion at position N37 or a position corresponding thereto.

4. The composition of claim 3, wherein the amino acid at position N37 or a position corresponding thereto is a hydrophilic amino acid that is polar and neutrally charged.

5. The composition of claim 4, wherein the polar, neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S), and threonine (T).

6. 6. The composition of claim 5, wherein the polar, neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

7. The composition of claim 6, wherein the polar, neutrally charged hydrophilic amino acid is glutamine (Q).

8. The composition of claim 1 , wherein the humanized single domain antibody is a variable fragment of a heavy chain antibody (VHH).

9. The composition of claim 8, wherein the VHH is specific for human serum albumin.

10. The composition of claim 8 or 9, wherein the VHH comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO: 3, and / or comprises three CDRs selected from (i) SEQ ID NOs: 12-14 or variants thereof, or (ii) SEQ ID NOs: 15-17 or variants thereof.

11. The composition of any one of claims 8 to 10, wherein the VHH further comprises substitutions or deletions in the framework regions relative to a non-humanized VHH.

12. The composition of any one of claims 1 to 11, wherein the recombinant human GM-CSF is linked to the single domain antibody via a linker.

13. The composition of claim 12 , wherein the linker is a flexible linker.

14. The composition of claim 13 , wherein the flexible linker consists essentially of glycine and serine residues.

15. 15. The composition of claim 13 or 14, wherein the flexible linker comprises at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60 amino acid residues.

16. The flexible linker is (Gly ４ Ser) ｎ wherein n is from about 1 to about 12.

17. The composition of claim 12, wherein the linker is a hinge-CH2-CH3 Fc domain.

18. The composition of claim 17, wherein the hinge-CH2-CH3 Fc domain is derived from human IgG.

19. 19. The composition of claim 18, wherein the hinge-CH2-CH3 Fc domain is derived from human IgG1, human IgG2, or human IgG4.

20. 13. The composition of claim 12, wherein the linker comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO:6; or an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5 having about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions.

21. 21. The composition of claim 20, wherein the linker comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO:

6.

22. The composition of any one of claims 1 to 21, wherein the VHH domain is located at the N-terminus of the fusion protein or chimeric protein.

23. The composition of any one of claims 1 to 21, wherein the GM-CSF is located at the N-terminus of the fusion protein or chimeric protein.

24. 24. The composition of any one of claims 1 to 23, wherein the fusion protein or chimeric protein comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; or an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

11.

25. 25. The composition of any one of claims 1 to 24, which binds to and / or activates the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R-α or CSF2R).

26. The composition of claim 25, wherein the GM-CSF-R-α is expressed on the surface of a cell.

27. The composition of claim 26 , wherein the cells are hematopoietic progenitor cells.

28. 28. The composition of claim 27, wherein the hematopoietic progenitor cells are immune cells.

29. 28. The composition of claim 27, wherein the hematopoietic progenitor cells are irradiated.

30. 30. The composition of any one of claims 1 to 29, which binds to human, mouse, and rhesus serum albumin.

31. The composition of claim 30, which binds to human serum albumin (HSA).

32. The composition of claim 31 , wherein the HSA is present in human plasma.

33. The composition of claim 32, wherein the composition does not substantially compete with the binding of HSA to the neonatal Fc receptor (FcRn).

34. The composition of any one of claims 1 to 33, wherein the fusion protein or chimeric protein is soluble.

35. The composition of any one of claims 1 to 34, wherein the fusion protein or chimeric protein exhibits substantially similar functionality to sargramostim.

36. The composition of claims 1 to 35, wherein the recombinant human GM-CSF comprises multiple molecular forms.

37. 37. The composition of claim 36, wherein the molecular form is selected from non-glycosylated, O-glycosylated, N-glycosylated, and N+O glycosylated forms.

38. The composition of any one of claims 1 to 37, wherein the recombinant human GM-CSF is substantially free of hypermannosylated forms.

39. A nucleic acid molecule encoding the fusion protein or chimeric protein of any one of claims 1 to 38, or a fragment thereof.

40. 40. The nucleic acid of claim 39, wherein the nucleic acid molecule has a codon-optimized sequence or a portion thereof.

41. 41. A human host cell expressing a nucleic acid molecule according to claim 39 or 40.

42. 42. The host cell of claim 41, which is a Chinese Hamster Ovary (CHO) cell.

43. A non-human host cell expressing a nucleic acid molecule according to claim 39 or 40.

44. 44. The non-human host cell of claim 43 which is a yeast cell.

45. 45. The non-human host cell of claim 44, wherein the yeast cell is a non-methylotrophic yeast cell.

46. 46. ​​The non-human host cell of claim 45, which is a Saccharomyces cerevisiae cell.

47. A pharmaceutical composition comprising the composition of any one of claims 1 to 38 and a pharma- ceutically acceptable excipient or carrier.

48. 48. A method for treating a patient or subject undergoing or having undergone cancer treatment, or a patient or subject undergoing or having undergone a bone marrow transplant, or a patient or subject acutely exposed to myelosuppressive amounts of radiation (e.g., H-ARS), and / or a patient or subject with Radiation Combined Injury (RCI), comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 47.

49. 49. The method of claim 48, wherein the patient is treated by modulating the clonal expansion, survival, differentiation, and activation state of hematopoietic progenitor cells.

50. 49. The method of claim 48, wherein the patient is treated by modulating the myelomonocytic cell lineage by promoting proliferation of megakaryocyte and erythroid progenitor cells.

51. 49. The method of claim 48, wherein the patient is treated by modulating hematopoietic progenitor cells by stimulating survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells.

52. 49. The method of claim 48, wherein the patient is treated by modulating hematopoietic progenitor cells by stimulating survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells following bone marrow transplantation.

53. Administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 47; or 48. A method of treatment comprising contacting a cell with an effective amount of the pharmaceutical composition of claim 47 and administering a therapeutically effective amount of said cell, The treatment, Promoting neutrophil recovery and / or reducing the incidence of infections following induction chemotherapy; Mobilizing hematopoietic progenitor cells into peripheral blood for harvesting by leukapheresis and transplantation; Promoting bone marrow reconstitution following autologous or allogeneic bone marrow progenitor cell or peripheral blood progenitor cell transplantation; To treat delayed neutrophil recovery or engraftment failure after autologous or allogeneic bone marrow transplantation; and / or Treating hematopoietic acute radiation syndrome (H-ARS); Treating radiation injury (RCI) Treating neurodegenerative diseases, Treating nerve cuts, Treating traumatic brain injury, Treating lung infections, Treating an autoimmune disease (optionally Crohn's disease or IBD); Treating sepsis / septicemia; Treating invasive fungal infections, Treating multiple organ dysfunction syndrome (MODS); Treat peripheral artery disease / peripheral arterial disease / cardiovascular disease; Treating oncology indications, A method of treatment for promoting wound healing and / or treating skin disorders.

54. 48. A method of treating a coronavirus infection comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

55. 55. The method of claim 54, wherein the coronavirus is a betacoronavirus, 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.

56. 56. The method of claim 55, wherein the coronavirus is an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

57. 57. The method of claim 56, wherein the coronavirus is SARS-CoV-2.

58. 57. The method of claim 56, wherein the patient is suffering from COVID-19.

59. 48. A method of treating an influenza virus infection, optionally selected from types A, B, C, and D, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

60. 48. A method for treating viral hemorrhagic fever, optionally caused by a group of viruses selected from arenaviruses, flaviviruses, filoviruses, hantaviruses, nairoviruses, fenuviruses, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

61. 48. A method for treating a pulmonary infection, wherein the pulmonary infection is pneumonic plague caused by infection with Yersinia pestis, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

62. 62. The method of any one of claims 54-61, wherein the patient is suffering from one or more of the following: fever, cough, shortness of breath, diarrhea, upper respiratory tract symptoms, lower respiratory tract symptoms, pneumonia, and acute respiratory syndrome.

63. 63. The method of any one of claims 54 to 62, wherein the patient is hypoxic.

64. 64. The method of any one of claims 54 to 63, wherein the patient is suffering from respiratory distress.

65. 65. The method of any one of claims 54 to 64, which prevents or reduces the development of Acute Respiratory Distress Syndrome (ARDS) in said patient.

66. 66. The method of any one of claims 54 to 65, which improves oxygenation in the patient.

67. 67. The method of any one of claims 54 to 66, which prevents or alleviates the transition from respiratory distress to cytokine imbalance in said patient.

68. 68. The method of any one of claims 54 to 67, which reverses or prevents a cytokine storm.

69. 69. The method of claim 68, which reverses or prevents a cytokine storm in the lungs or the whole body.

70. 70. The method of claim 68 or 69, wherein the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis.

71. 71. The method of claim 70, which reverses or prevents excessive production of one or more inflammatory cytokines.

72. 72. The method of claim 71, wherein the proinflammatory cytokine is one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

73. 73. The method of any one of claims 54 to 72, which causes a reduction in the viral or bacterial load in the patient compared to before treatment.

74. 48. A method of treating and / or ameliorating symptoms caused by exposure to a chemical weapon comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

75. 75. The method of claim 74, wherein said chemical weapon is sulfur mustard gas (SM / HD) and / or chlorine gas.

76. 76. The method of claim 74 or 75, wherein symptoms of exposure in the patient are characterized by bone marrow suppression.

77. 77. The method of any one of claims 74-76, wherein the composition causes amelioration of symptoms of chemical exposure, including bone marrow suppression.

78. 48. A method for treating and / or ameliorating symptoms caused by a neurodegenerative disease, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

79. 79. The method of claim 78, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), dementia with Lewy bodies, Parkinson's disease dementia, epilepsy, stroke, Huntington's chorea, cerebral hypoxia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and peripheral neuropathy.

80. 80. The method of claim 78 or 79, wherein the neurodegenerative disease is characterized by oxidative stress, loss of neurite integrity, apoptosis, neuronal cell loss, or / and an inflammatory response.

81. The method of any one of claims 78 to 80, wherein the neurodegenerative disease is associated with cognitive impairment.

82. 82. The method of any one of claims 78 to 81, wherein the composition induces a disease-modifying response.

83. 83. The method of any one of claims 78 to 82, wherein the composition slows down the decline in cognitive function temporarily or permanently.

84. The method of any one of claims 78 to 83, wherein the composition causes amelioration of symptoms of a neurodegenerative disease.

85. 85. The method of any one of claims 78 to 84, wherein the composition delays the onset and / or development of a neurodegenerative disease.

86. 48. A method of treating an autoimmune disease comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47, wherein the autoimmune disease is optionally selected from Crohn's disease and / or ulcerative colitis (UC) and / or irritable bowel disease (IBD).

87. 87. The method of claim 86, wherein the composition restores a balance between effector and regulatory immune function.

88. A method for treating and / or ameliorating symptoms caused by caspase recruitment domain family member 9 (CARD9) deficiency, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition described in claim 47.

89. 89. The method of claim 88 for treating an invasive fungal infection.

90. 90. The method of claim 89, wherein the invasive fungal infection is selected from candidiasis, mucormycosis, invasive aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, basidiobolus, conidiobolus, cryptococcal meningitis, and histoplasmosis.

91. 91. The method of claim 90, wherein the candidiasis is esophageal candidiasis, oropharyngeal candidiasis, invasive candidiasis and / or candidemia.

92. 91. The method of claim 90, wherein the mucormycosis is cutaneous mucormycosis.

93. The method of any one of claims 88 to 92, wherein the composition is used as an adjunct to antifungal therapy.

94. 94. The method of any one of claims 88 to 93, wherein the composition aids in the decrease and / or reduction of fungal burden in various tissues.

95. 48. A method of treating and / or promoting wound healing and / or skin disorders comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

96. 96. The method of claim 95, wherein the chronic wound is optionally selected from leprosy, leg ulcers, and / or non-healing wounds due to various other causes.

97. 97. The method of claim 96, which promotes tissue remodeling and regeneration.

98. 48. A method of treating cancer comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

99. 99. The method of claim 98, wherein the composition causes immunomodulation or immunoactivation of an immune response against tumor cells.

100. 100. The method of claim 98 or 99, wherein the composition enhances or restores the activity or activation of one or more cells selected from the group consisting of T cells, T helper cells, cytotoxic T cells, dendritic cells, NK cells, macrophages, anti-tumor macrophages, and B cells.

101. The method of claims 98-100, wherein the composition treats, ameliorates and / or prevents cancer proliferation, survival, metastasis, epithelial-mesenchymal transition, immune evasion, or recurrence.

102. 48. A method of treating sepsis or septicemia comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

103. The method of claims 98-101, wherein the composition balances and / or modulates inflammation and the acute phase response, reducing morbidity and mortality.

104. 48. A method of treating immune thrombocytopenia (ITP) or low platelet count comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 47.

105. 104. The method of claim 103, wherein the composition boosts or enhances platelet cell count and / or reverses thrombocytopenia.

106. A composition according to any one of claims 1 to 38, which facilitates delivery of recombinant GM-CSF through endothelial capillaries (blood-brain barrier, BBB) for central nervous system (CNS) treatment.

107. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein is produced by transfection of a mammalian cell, optionally a CHO cell, and wherein the fusion protein or chimeric protein exhibits an increased half-life compared to sargramostim.

108. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein exhibits enhanced pharmacokinetics compared to sargramostim.

109. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein exhibits substantially similar functionality to sargramostim.

110. 39. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein significantly increases the level, cell count, and / or amount of neutrophils, monocytes, eosinophils, total white blood cells, and platelets.

111. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein significantly increases platelet levels, cell counts and / or mass.

112. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein is produced by transfection of a mammalian cell, optionally a CHO cell, and wherein the chimeric protein exhibits improved pharmacodynamics compared to sargramostim.

113. The composition of any one of claims 1 to 38, wherein the fusion protein or chimeric protein is produced by transfection of a mammalian cell, optionally a CHO cell, and wherein the fusion protein or chimeric protein exhibits similar function and improved pharmacokinetics and pharmacodynamics compared to sargramostim.

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