Long-acting granulocyte macrophage-colony stimulating factors

Abstract

The colony stimulating factor (CSF) refers to a family of four glycoproteins that control and coordinate cellular production of broadly dispersed bone marrow cell deposits. GM-CSF is a blood growth factor that regulates the production, migration, proliferation, differentiation and function of hematopoietic cells. The present disclosure relates generally to compositions and methods related to long acting granulocyte-macrophage colony stimulating factor (GM-CSF) with improved pharmacokinetics.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 329,039, filed on April 8, 2022, the entire contents of which are hereby incorporated by reference for all purposes. Technical Field

[0003] The present disclosure relates, inter alia, to compositions and methods relating to granulocyte-macrophage colony stimulating factor (GM-CSF) that is long-acting and has improved pharmacokinetics.

[0004] Description of the text file submitted electronically

[0005] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable format copy of the Sequence Listing (File Name: "PNR-009PC 127114-5009-PC Sequence Listing.xml"; Recording Date: March 23, 2023; File Size: 20,480 bytes). Background Art

[0006] Colony stimulating factor CSF refers to a family of four glycoproteins that control and coordinate the production of cells in widely dispersed bone marrow cell deposits. These include: granulocyte-macrophage CSF (GM-CSF), granulocyte colony CSF (G-CSF), macrophage colony CSF (M-CSF) and multipotent colony stimulating factor (IL-3). These lymphokines can induce progenitor cells present in the bone marrow to differentiate into specific types of mature blood cells. The specific type of mature blood cells produced by progenitor cells depends on the type of CSF present. See Metcalf D. Cancer Immunol Res. 2013, 1 (6): 351-356.

[0007] GM-CSF is a blood growth factor that regulates the production, migration, proliferation, differentiation, and function of hematopoietic cells. In response to inflammatory stimuli, various cell types, including T lymphocytes, macrophages, fibroblasts, and endothelial cells, release GM-CSF. GM-CSF then activates and enhances the production and survival of neutrophils, eosinophils, and macrophages. Native 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 only at picomolar concentrations (10 -10 Up to 10 -12M) is present in circulating blood. 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.

[0008] Human GM-CSF (hGM-CSF) is synthesized as a 144 amino acid residue precursor protein with a 17 amino acid signal peptide. This precursor protein is processed to produce a 127 amino acid mature protein with a predicted molecular mass of 14.4 kDa. It has two disulfide bonds that migrate as a broad band of 15-30 kDa due to glycosylation and sialylation. The N-linked glycans are located at Asn 27 and Asn 37 At Ser5, Ser7, Ser9 and Ser10, and multiple potential sites for O-linked glycosylation exist, but the extent of the 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.

[0009] Recombinant human granulocyte-macrophage colony stimulating factor (rhu GM-CSF) has been approved by the FDA for use in combination with chemotherapy to treat neutropenia, dysbiosis, and malignancies such as leukemia. Clinically, GM-CSF used to treat neutropenia and aplastic anemia after chemotherapy greatly reduces the risk of infection associated with bone marrow transplantation. Its utility in the treatment of myeloid leukemia and as a vaccine adjuvant has also been well established. See DorrRT.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.

[0010] Although there are five classes of heterologous protein production platforms, including bacteria, yeast, plants, insect cells, and mammalian cells, more than 50% of currently marketed biopharmaceuticals are produced in mammalian cell lines. This is partly due to the inability of the remaining four classes to modify glycoproteins with human-like oligosaccharides. This is important because protein-bound glycans affect circulation half-life, tissue distribution, biological activity, and immunogenicity.

[0011] GM-CSF expression system affects the pharmacokinetic properties, biological activity and clinical toxicity of GM-CSF. In clinical practice, GM-CSF has been produced in Chinese hamster ovary cells (CHO-GM, regramostim), Escherichia coli (E. coli-GM, molgramostim) or yeast (yeast-GM, sargramostim). See DorrRT.ClinTherapeutics.1993.15(1):19-29; WalshG.Nat Biotechnol.2006,24:769-776; JacobPP et al.Nat Protoc.2009,4:58-70; JacobPP et al.Microbial Cell Factories 2010,9:93; WalshG.Nat Biotechnol.2018,36(12):1136-1145. In the manufacture of GM-CSF, there are four major GM-CSF isoforms / species: "highly glycosylated" (about 50 kDa), N- and N-+O-glycosylated (about 20 kDa), O-glycosylated (about 16 kDa), and non-glycosylated (about 15 kDa) species. Glycosylated GM-CSF is obtained via recombinant techniques using yeast (sargramostim) or Chinese hamster ovary (CHO) cell (relastostim) technology, which produces a complex mixture of glycoforms. Human cell-derived GM-CSF is also heterogeneously glycosylated, however, when manufactured in E. coli, GM-CSF remains non-glycosylated. Glycan heterogeneity reflects the lack of specificity of 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.

[0012] N-glycosylation occurs when the glycan is added to the nitrogen of the asparagine (Asn) or arginine (Arg) side chain, while O-glycosylation occurs when the glycan is added to the hydroxyl oxygen side chain of a serine (Ser) or threonine (Thr) or tyrosine (Tyr) amino acid. Asparagine residues generally require a consensus sequence of Asn-Xxx-Ser / Thr / Cys, where Xxx can be any amino acid except proline, for N-glycosylation. The rhu GM-CSF protein sequence of sargramostim contains two consensus sequences, Asn-Leu-Ser ( NLS ) and Asn-Glu-Thr( NET ), wherein the protein can potentially be glycosylated. During the manufacturing process of sargramostim, two N-glycosylation sites, Asn 27 and Asn 37Glycosylated in some GM-CSF species present in the fermenter. 39 (or Ser 39 or Cys 39 ) residue is Asn 27 or Asn 37 Species with N-oligosaccharides attached at asparagine residue 37 are called "hyperglycosylated" species (30-100 kDa) because these glycoforms contain up to several hundred mannose residues. Hypermannosylation occurs only at N37.

[0013] The final product of sargramostim (LEUKINE) consists of N-glycosylated and O-glycosylated glycoforms together with non-glycosylated glycoforms. Thus, sargramostim has heterogeneity in its glycoform profile, which has been very consistent throughout its licensing history. Nearly half of the GM-CSF protein in sargramostim is non-glycosylated, and slightly less than one-third is fully N-glycosylated. Most, if not all, of the N-glycosylated species are also O-glycosylated. The highly branched N-linked oligosaccharide structure covers most of the molecule, including the H1 and H5 α-helices, without obstructing the receptor binding site, as well as some H6 α-helices and the C-terminal random coil. Although the carbohydrate composition of recombinant GM-CSF differs based on the cell source, glycosylation is not required for receptor binding or in vitro and in vivo biological activity. Yeast rhu GM-CSF and non-glycosylated E. coli-derived rhu GM-CSF have comparable specific activities measured by both receptor affinity and cell proliferation specific activity. See Urdal and Park, 1988; Metcalf D. Cancer 1990: 65: 2185-2195.

[0014] Endogenous proteins in the human body are degraded at different rates in vivo, and the rates may vary widely due to their functions. For example, hemoglobin lasts the entire life cycle of red blood cells and histones have a half-life measured in years, while other proteins such as ornithine decarboxylase (11min half-life) have very fast degradation rates. See Thomas E. Creighton (1993). "Chapter 10-Degradation". Proteins: Structures and Molecular Properties (2nd edition). W. H. Freeman and Company. Pages 463-473. ISBN 0-7167-2317-4. In eukaryotes, most 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. UPP plays a central role in the intracellular homeostasis in the processing and degradation of proteins, including those proteins that regulate cell cycle progression, apoptosis and DNA repair. See Heinemeyer W et al. J Biol Chem. 1997. 272: 25200-25209. Lysosomes also remove unwanted proteins in the cytoplasm and outside the cell. Materials from outside the cell are taken up by endocytosis, while materials from inside the cell are digested by autophagy. See Settembre C et al. Nature Reviews Molecular Cell Biology. 2013. 14 (5): 283-96. Studies have shown that GM-CSF has a half-life that can be extended from 50 to 85 min. See Cebon et al. Blood. 1988. 72: 1340-1347; Stagg et al. 2004. The in vivo biological activity of naturally occurring and recombinant GM-CSF depends largely on their bioavailability. See Dorr RT. Clinical Therapeutics / Vol. 15, No. 1, 1993.

[0015] Glycosylation is reported to stabilize a variety of proteins. Studies have found that some charged terminal glycans (e.g., sialic acid) are essential for regulating the circulating half-life of glycoproteins. There are also reports that glycosylation is essential for maintaining protein conformational stability against pH denaturation, such as 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 engineered into proteins, such as by glycation of penicillin G acylase. The half-life of the glycated form of this protein increased 13-fold at pH 3 and 7-fold at pH 10 when compared to the non-glycosylated protein. See Masarova J, Biotechnol Appl Biochem. 2001; 34 (Part 2): 127-133. Studies have also shown that both the degree of glycosylation and the size of glycans can increase the inactivation half-life of proteins, but as the size of glycans increases, the degree of kinetic stability increases significantly. See Solá 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.

[0016] The bioavailability of therapeutic proteins (biotherapeutics or bio) in humans may also be affected by the route of drug administration. See Dorr RT, Clinical Therapeutics / Volume 15, Issue 1, 1993. After intravenous (IV) administration, almost all therapeutic proteins are immediately available, but then over time, they may be degraded by proteases present in the plasma. In some cases, subcutaneous (SC) administration prolongs the exposure of proteins with short elimination half-lives by maintaining high plasma concentrations for a longer period of time, and may be better tolerated than IV administration. Therefore, the absorption of biotherapeutics after SC administration may be affected by its fate in the extracellular matrix (ECM), including the possibility of incomplete bioavailability. See Hale G et al. Blood. 2004. 104 (4): 948-955. The transport of different isoforms present in sargramostim to the systemic circulation may be beneficial to different pathways because of their different molecular sizes.

[0017] GM-CSF expression systems can affect pharmacokinetic parameters, among which the degree of glycosylation affects GM-CSF half-life, distribution and elimination. The relationship between the degree of glycosylation of GM-CSF and its distribution, clearance and activity was reported using a rat model. Studies have found that the specific activity of GM-CSF measured in vitro is significantly reduced in the largest, most fully glycosylated form of the protein relative to smaller, less glycosylated molecules. After a single intravenous bolus injection, the effective half-life of GM-CSF in the rat bloodstream was shown to be significantly increased by the addition of N-linked carbohydrates. The clearance of GM-CSF in rats follows biphasic kinetics, and carbohydrate modification prolongs the first phase or c phase. 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 affect pharmacokinetic parameters is to conjugate the target protein (such as GM-CSF) to albumin. This can take advantage of the long half-life and targeting ability of albumin to enhance the biological stability of the target protein. See Chuang YM et al. Cell Mol Immunol. 2020.

[0018] Studies have also shown that anti-serum albumin domain antibodies extend the half-life of short-lived drugs. See Holt LJ et al. Protein Eng Des Sel. 2008. 21 (5): 283-8. However, conventional antibodies are large in size, which can hinder the delivery of target proteins or drugs to cells in vivo. Nanobodies (15kDa) and human heavy chain antibodies (75kDa) based on nanobodies can overcome these limitations. Camelids naturally produce antibodies consisting only of heavy chains, in which the target recognition module consists of a single variable domain (VHH or Nb). The favorable features of nanobodies include their 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 (such as anti-tumor nanobodies) can be extended by fusing one or more anti-tumor nanobodies with albumin-specific nanobodies. 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 increasing the half-life and circulation time of GM-CSF-based therapies could reduce the dose and simplify the regimen of drugs administered to patients. This could have a positive impact on the quality of patient care, patient compliance, and reduced healthcare costs.

[0019] There remains a need for GM-CSF molecules with improved pharmacokinetics, increased half-life and increased circulation time. Summary of the invention

[0020] Thus, the present disclosure relates to long-acting forms of GM-CSF that, among other things, provide improved pharmacokinetics and pharmacodynamics relative to wild-type GM-CSF or sargramostim.

[0021] In various aspects, a fusion or chimeric protein is provided, comprising a recombinant human GM-CSF protein, a linker, and a humanized single domain antibody.

[0022] In various aspects, the recombinant human GM-CSF protein of the composition of the present invention comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 1 or SEQ ID NO: 2 and optionally having a substitution or deletion at position N37 or its corresponding position, for example, the amino acid at position N37 or its corresponding position can be substituted with a polar and neutrally charged hydrophilic amino acid, such as glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C).

[0023] In various aspects, a humanized single domain antibody of a composition of the invention comprises a heavy chain having three CDRs (eg, as defined by IMGT nomenclature or Kabat nomenclature).

[0024] IMGT nomenclature:

[0025] CDR-H1:GETLDYYA(SEQ ID NO:12)

[0026] CDR-H2:IASSGGST(SEQ ID NO:13)

[0027] CDR-H3:AAAVLECRTVVRGYDY(SEQ ID NO:14)

[0028] Kabat CDR Definition:

[0029] CDR-H1:ETLDYYAIG(SEQ ID NO:15)

[0030] CDR-H2:IASSGGSTNYADSVKG(SEQ ID NO:16)

[0031] CDR-H3: AVLECRTVVRGYDY (SEQ ID NO: 17).

[0032] In various aspects, the humanized single domain antibody of the composition of the present invention comprises a heavy chain having three CDRs of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or a variant having one or more substitutions or deletions (e.g., 1, or 2, or 3, or 4). In various aspects, the humanized single domain antibody of the composition of the present invention comprises a heavy chain having three CDRs of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, or a variant having one or more substitutions or deletions (e.g., 1, or 2, or 3, or 4). In various aspects, the humanized single domain antibody of the composition of the present 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 with SEQ ID NO: 3, and having substitutions or deletions within the framework of a non-human camelid antibody to enable humanization of the non-human antibody. The CDRs are defined as in the IMGT nomenclature. CDRs may also be defined using Kabat nomenclature.

[0033] IMGT nomenclature:

[0034] CDR-H1:GETLDYYA(SEQ ID NO:12)

[0035] CDR-H2:IASSGGST(SEQ ID NO:13)

[0036] CDR-H3:AAAVLECRTVVRGYDY(SEQ ID NO:14)

[0037] Kabat CDR Definition:

[0038] CDR-H1:ETLDYYAIG(SEQ ID NO:15)

[0039] CDR-H2:IASSGGSTNYADSVKG(SEQ ID NO:16)

[0040] CDR-H3:AVLECRTVVRGYDY(SEQ ID NO:17)

[0041] In various aspects, the fusion or chimeric protein (protein comprising recombinant human GM-CSF protein) is fused to the humanized single domain antibody via a linker.

[0042] In an embodiment, the composition of the invention comprises a plurality of molecular forms, such as non-glycosylated, O-glycosylated, N-glycosylated and / or N+O glycosylated forms. In an embodiment, the fusion or chimeric protein of the invention is substantially free of hyperglycosylated, such as hypermannosylated forms.

[0043] In an embodiment, a nucleic acid molecule encoding a fusion or chimeric protein of the present invention (e.g., a codon-optimized sequence) is also provided. In an embodiment, a human host cell expressing a nucleic acid molecule encoding a fusion or chimeric protein of the present invention is also provided, optionally a Chinese hamster ovary (CHO) cell. In an embodiment, a pharmaceutical composition is also provided, comprising a fusion or chimeric protein of the present invention and a pharmaceutically acceptable excipient or carrier.

[0044] In various aspects, a method of treating a patient or subject who is undergoing or has undergone cancer therapy or is undergoing or has undergone a bone marrow transplant or has been acutely exposed to a myelosuppressive dose of radiation (e.g., acute radiation sickness or acute radiation syndrome (ARS)) and / or has radiation-compounded injury (RCI) is provided; the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition described herein.

[0045] In various aspects, 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 disease dementia epilepsy, stroke, Huntington's disease, cerebral hypoxia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and peripheral neuropathy) is provided, and a subject in need thereof has been treated with a fusion or chimeric protein described herein or a pharmaceutical composition thereof.

[0046] In various aspects, a method of treating a fungal infection (such as but not limited to an invasive fungal infection optionally selected from candidiasis and / or mucormycosis infection) is provided, and a subject in need thereof has been treated with a fusion or chimeric protein described herein or a pharmaceutical composition thereof.

[0047] In various aspects, a method of treating an autoimmune disease is provided, wherein the autoimmune disease is optionally selected from Crohn's disease and / or ulcerative colitis (UC), irritable bowel disease (IBD) and / or other GI inflammatory diseases, wherein a patient or subject in need thereof has been treated with a fusion or chimeric protein described herein or a pharmaceutical composition thereof.

[0048] In embodiments, the fusion or chimeric protein of the invention is functionally similar to sargramostim. In embodiments, the fusion or chimeric protein has enhanced pharmacokinetics as compared to recombinant human GM-CSF. In embodiments, the fusion or chimeric protein has enhanced half-life as compared to recombinant human GM-CSF. In embodiments, the fusion or chimeric protein has enhanced pharmacodynamics as compared to recombinant human GM-CSF. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A The structural representation of heavy chain IgG (hcIgG) is illustrated.

[0050] Figure 1B A yeast surface display system for screening a library of antigen-binding scaffolds is shown. VHH is fused to the N-terminus of Aga2p via its C-terminus. Surface expression can be detected using a fluorescently labeled antibody that binds to the Myc tag.

[0051] Figure 1C Figures showing VHHs promoting half-life extension of Fab fragments in a murine model are illustrated. Different lines on the figure illustrate different constructs. Bi-340-352 is an anti-albumin VHH half-life extension molecule as a fusion partner to the Fab fragment. Bi-340-356 is an unrelated VHH fusion fused to the same Fab fragment that did not show half-life extension.

[0052] Figure 2 Four kinds of constructs cloned and transiently expressed in CHO cells with or without secretion leader sequences are illustrated. The secretion leader sequences of all 4 constructs are identical. The 4 constructs include: VHH-G4Sx4 joint-GMCSF, VHH-hinge joint-GMCSF, GMCSF-hinge joint-VHH and GM-CSF-G4Sx4 joint-VHH. The figure also illustrates VHH-G4Ax4 joint-GM-CSF with or without secretion leader sequences.

[0053] Figure 3A-B shows a graph of the results of the TF-1 assay of various fusion or chimeric proteins, CHO produced GM-CSF (ATUM CHO N37Q) and LEUKINE / QCRS-GMCSF (shown as separate graph lines) with and without human serum albumin (HSA). BDS LEUKINE / QCRS GMCSF refers to wild type GM-CSF, i.e., SEQ ID NO:2, without T39A or N37Q amino acid substitutions within the sequence. CHO N37Q is the CHO produced GMCSF variant used in the VH H constructs. 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 40ug / ml (physiological concentration in blood) of HSA to the cell assay.

[0054] Figure 4A Exemplary spectra for verifying the heterogeneity of N-terminal cleavage and glycosylation of VHH-G4Sx4-GMCSF variants (VHH-G4Sx4-GMCSF*) are illustrated. High-resolution liquid chromatography-mass spectrometry (LC-MS) (Vanquish UPLC QExactive, Thermo) was used to determine the complete molecular weight and glycan spectrum. The figure shows the glycan analysis performed by mass spectrometry, in particular the glycan occupancy of 6 stable CHO clones constituting VHH-G4A GMCSF*. 100 μg of sample was diluted to 2 mg / mL with deionized water and then injected into LC-MS. The data was analyzed and deconvoluted by BioPharmaFinder software to obtain the complete protein molecular mass. Mass spectrometry also shows that the known glycosylation sites in GMCSF: Ser7 Ser10 and N27 are also glycosylated throughout the VHH-G4Sx4-GMCSF variants, and are similar to the GMCSF* produced by CHO used in the fusion protein.

[0055] Figure 4B A graph depicting the results of the TF-1 assay is shown. In the TF-1 cell based assay, VHH-G4A GMCSF* produced by six stable CHO clones was analyzed.

[0056] Figure 5A-C shows a diagram illustrating the evaluation of the binding of VHH to three types of albumin (including human, murine and rhesus monkey) by surface plasmon resonance (SPR). To evaluate binding, VHH was cloned onto human IgG1 Fc and hinge for ease of purification and analysis. The binding affinity of the VHH-Fc construct was determined by BLI using Octet 384 (Pall Life Sciences). The VHH-Fc construct was loaded onto the AHC sensor and then exposed to a monomer target (human, mouse or rhesus monkey albumin) in solution to determine association and dissociation. In the case of pH evaluation, the binding reaction and dissociation buffers were at the described pH. Data acquisition was performed at 30°C.

[0057] Fig. 6A -D shows a graph illustrating the evaluation of VHH binding to human albumin at different pH at 30°C by surface plasmon resonance (SPR). The graph shows response units on the Y axis (binding of target to VHH-Fc on the chip sensor) and time on the X axis. The data (time and response units) were analyzed using ForteBio data analysis software.

[0058] Fig. 7A -C shows the half-life (t) of the fusion or chimeric protein VHH-GA-GMCSF* compared with CHO-produced GM-CSF and LEUKINE after intravenous injection of 10 ug / Kg of the listed forms of GMCSF. 1 / 2 ) or pharmacokinetic plots. A single rhesus monkey was injected intravenously with 10 ug / Kg of the listed forms of GMCSF. Blood was drawn at 12 min, 30 min, 1, 2, 4, 8, 24, 48 and 72 hours after injection and analyzed for cell content and the concentration of constructs in the blood was determined by construct-specific ELISA. VHH-GA-GMCSF* shows approximately 17-fold higher t than the Leukine or CHO-produced GMCSF forms used in the VHH-GA-GMCSF constructs. 1 / 2 The CHO control is a variant of GMCSF used on VHH constructs made in CHO cells (GMCSF R23L and N37Q) to maintain similar glycosylation patterns. The yeast control is LEUKINE.

[0059] Fig. 8A-E shows a graph showing the number of neutrophils, monocytes, eosinophils, total WBCs and platelets determined after intravenous (IV) injection of fusion or chimeric protein constructs as compared to CHO and LEUKINE (yeast grown GM-CSF). The CHO control is a variant of GMCSF used on VHH constructs made in CHO cells (GMCSF R23L and N37Q) to maintain a similar glycosylation pattern. DETAILED DESCRIPTION

[0060] The present disclosure is based, in part, on fusion or chimeric proteins comprising a recombinant human GM-CSF protein, a linker, and a humanized single domain antibody, wherein the composition produces a functional GM-CSF having enhanced half-life, pharmacokinetics, and pharmacodynamics.

[0061] Composition of GM-CSF

[0062] In one aspect, engineered fusion or chimeric proteins are provided.

[0063] In embodiments, engineered GM-CSFs useful in practicing the present disclosure include any pharmaceutically safe and effective GM-CSF, or any derivative thereof having the biological activity of GM-CSF and optionally having substitutions and / or deletions of the present invention.

[0064] In one embodiment, the engineered GM-CSF used to practice the subject methods is derived from recombinant human GM-CSF (rhuGM-CSF), such as sargramostim (LEUKINE). Sargramostim is a biosynthetic, yeast-derived, recombinant human GM-CSF, a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF by having a leucine instead of an arginine at position 23. Other natural and synthetic GM-CSFs and derivatives thereof that have the biological activity of native human GM-CSF can also be used in the practice of the present disclosure.

[0065] In an embodiment, the recombinant human GM-CSF molecule of the invention is glycosylated. In an embodiment, the recombinant human GM-CSF molecule of the invention comprises one or more substitutions and / or deletions that affect the glycosylation of GM-CSF.

[0066] Without wishing to be bound by theory, the degree of glycosylation of biosynthetic GM-CSF appears to affect half-life, distribution, and elimination (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).

[0067] In one aspect, a recombinant human GM-CSF protein is provided, comprising an amino acid sequence that is 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% identical, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 2. In one aspect, a recombinant human GM-CSF protein is provided, comprising an amino acid sequence that is 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% identical, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 2 and has a substitution or deletion at position N37 or a position corresponding thereto.

[0068] In an embodiment, the amino acid at position N37 of recombinant human GM-CSF or its corresponding position is a polar and neutrally charged hydrophilic amino acid. In an embodiment, the polar and neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In an embodiment, the polar and neutrally charged hydrophilic amino acid is glutamine (Q). In an embodiment, the polar and neutrally charged hydrophilic amino acid is selected from glutamine (Q), serine (S) and threonine (T).

[0069] In an embodiment, a recombinant human GM-CSF protein is provided, 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 having one or more of N37 deletion, N37Q, N37S, N37T, N37P, and N37C.

[0070] In an embodiment, the recombinant human GM-CSF has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof having about 90%, or about 93%, or about 95%, or about 97%, or about 98% identity thereto and optionally having substitutions and / or deletions of the present invention.

[0071] In an embodiment, the GM-CSF is one of molaspostim, sargramostim and relastostim, optionally with substitutions and / or deletions of the invention.

[0072] Without wishing to be bound by theory, the core of hGM-CSF consists of four helices arranged at an angle. Crystal structure and mutagenesis analysis of rhGM-CSF (Rozwarski DA et al., Proteins 26: 304-13, 1996) show that, in addition to the non-polar side chains in the protein core, 10 buried hydrogen-bonded residues involve intramolecular hydrogen bonding with backbone atoms that are more conservative than residues hydrogen-bonded to other side chain atoms; 24 solvation sites were observed at equivalent positions in the two molecules in the asymmetric unit, and the strongest of these were located in the gaps between secondary structure elements. Two surface clusters of hydrophobic side chains are located near the expected receptor binding region. Mutagenesis of residues on the helix A / helix C surface confirmed the importance of certain Glu, Gly and Gln residues. Therefore, these residues will not be substituted in the functional substitution variants of hGM-CSF used in the present disclosure, and these helices will be retained in the functional fragments or deletion variants of hGM-CSF used in the present disclosure.

[0073] Furthermore, in embodiments, one of ordinary skill may refer to the structural information of UniProtKB entry P04141 to inform the identity of the variant.

[0074] The N-terminal helix of hGM-CSF controls the high affinity binding to its receptor (Shanafelt AB et al., EMBO J10: 4105-12, 1991). The 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). The above studies identified the receptor binding determinant cluster in GM-CSF by locating a unique receptor binding domain on a series of human-mouse hybrid GM-CSF cytokines. The interaction of GM-CSF with the shared subunits of its high affinity receptor complex is controlled by a small part of the peptide chain. The presence of several key residues in the N-terminal α-helix is ​​sufficient to confer specificity to the interaction.

[0075] Thus, in embodiments, this information can inform the skilled artisan regarding acceptable changes in amino acid sequences.

[0076] In an embodiment, the amino acid mutation is an amino acid substitution, and may include conservative substitutions and / or non-conservative substitutions.

[0077] "Conservative substitutions" can be made, for example, based on the polarity, charge, size, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0078] As used herein, "conservative substitution" is defined as an amino acid exchanged by another amino acid listed in the same group of the six standard amino acid groups shown above. For example, Asp is exchanged by Glu to retain a negative charge in the polypeptide modified in this way. In addition, glycine and proline can replace each other based on their ability to destroy α-helices.

[0079] As used herein, "non-conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in a different group among the six standard amino acid groups (1) to (6) shown above.

[0080] In various embodiments, substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs in general).

[0081] The modification of the amino acid sequence can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described in, for example, 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.

[0082] Sugar type

[0083] In an embodiment, the fusion or chimeric protein of the invention contains a GM-CSF molecule comprising a plurality of molecular forms. In an embodiment, the molecular form is selected from the group consisting of non-glycosylated, O-glycosylated, N-glycosylated, and N+O glycosylated forms.

[0084] In embodiments, the fusion or chimeric protein is substantially free of hyperglycosylation, such as hypermannosylated forms. In embodiments, the recombinant human GM-CSF has about or less than about 10% of hypermannosylated forms after purification, or about or less than about 5% of hypermannosylated forms after purification, or about or less than about 3% of hypermannosylated forms after purification, or about or less than about 2% of hypermannosylated forms after purification, or about or less than about 1% of hypermannosylated forms after purification.

[0085] In an embodiment, the fusion or chimeric protein has less hypermannosylated forms than wild-type human GM-CSF and / or sargramostim when expressed and purified in the same manner. In an embodiment, 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 in the same manner. In embodiments, the amount of the hypermannosylated form can be detected as known in the art, such as, but not limited to, chromatographic methods (e.g., size resolution (e.g., by molecular weight and / or retention time in 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., a gel staining procedure based on the periodic acid-Schiff (PAS) reaction), affinity-based methods (such as using sugar-binding proteins (e.g., lectins)), enzyme-based methods, antibody-based methods, release assays (e.g., enzymatic cleavage or chemical removal or chemical derivatization of glycans), capillary electrophoresis, and eastern blot.

[0086] In embodiments, the fusion or chimeric proteins of the invention are amenable to a less complex purification than that used for sargramostim.

[0087] In an embodiment, the fusion or chimeric GM-CSF protein of the invention is resolved into three peaks when quantified by reverse phase high performance liquid chromatography (RP-HPLC). In an embodiment, the composition of the invention is resolved into three forms (aglycosylated form, O-linked glycoform, and O+N-linked glycoform), e.g., by HPLC, and lacks a hyperglycosylated form.

[0088] In embodiments, the fusion or chimeric GM-CSF proteins of the invention can be purified without the need for highly glycosylated species, without the need for one or more organic solvents (such as, but not limited to, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid, and / or N-propanol), and / or a reverse phase C4 HPLC column for the purpose of purification and / or removal of highly glycosylated peaks (aglycosylated forms, O-linked glycoforms, and O+N-linked glycoforms).

[0089] In an embodiment, the recombinant human GM-CSF of the present invention comprises more than one species (eg, glycoforms).

[0090] In an embodiment, the fusion or chimeric protein produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, is monomeric with heterogeneous glycoforms and has glycosylated and aglycosylated isoforms.

[0091] Humanized single domain antibody (VHH)

[0092] In various aspects, the humanized single domain antibody of the composition of the invention comprises an amino acid sequence that is 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% identical to SEQ ID NO: 3, and has substitutions or deletions within the non-human camelid antibody framework to enable humanization of the non-human antibody. The CDRs are defined as in the IMGT nomenclature. CDRs may also be defined using the Kabat nomenclature.

[0093] In various aspects, the humanized single domain antibody of the composition of the invention comprises an amino acid sequence that is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical to SEQ ID NO.3 and / or comprises three CDRs selected from (i) SEQ ID NO: 12-14, or variants thereof, or (ii) SEQ ID NO: 15-17, or variants thereof.

[0094] In an embodiment, the humanized single domain antibody (VHH) of the composition of the invention binds to human, mouse and rhesus serum albumin. In an embodiment, the humanized single domain antibody (VHH) of the composition of the invention binds to and / or is specific for human serum albumin (HSA). In an embodiment, the composition of the present disclosure binds to HSA, wherein HSA is present in human plasma.

[0095] In an embodiment, the compositions of the invention do not substantially compete with HSA for binding to the neonatal Fc receptor (FcRn).

[0096] In an embodiment, the compositions of the invention bind Protein A.

[0097] In an embodiment, when the fusion or chimeric protein of the present invention is soluble, the protein binding activity can be expressed as a dissociation constant (KD). Optionally, when the fusion or chimeric protein is a membrane antigen, the antigen binding activity can be expressed as an apparent dissociation constant (apparent KD).

[0098] In an embodiment, 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.

[0099] In 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 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.

[0100] In embodiments, the binding activity of the composition may be determined under different conditions. In a preferred embodiment, the measurement conditions are constant.

[0101] Connectors

[0102] In various aspects, the fusion or chimeric protein (protein comprising recombinant human GM-CSF protein) is linked and / or fused to the humanized single domain antibody via a linker.

[0103] In an embodiment, the linker of the composition of the invention is a flexible linker. In an embodiment, the flexible linker of the composition of the invention substantially comprises glycine and serine residues. In an embodiment, the flexible linker of the composition of the invention 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.

[0104] In one embodiment, the flexible linker of the composition of the present invention comprises (Gly 4 Ser) n , wherein n is from about 1 to about 12.

[0105] In one embodiment, the flexible linker of the composition of the present invention comprises (Gly 4 Ala) n , wherein n is from about 1 to about 12.

[0106] In one embodiment, the flexible linker of the composition of the present invention is a hinge-CH2-CH3 Fc domain. In an embodiment, the hinge-CH2-CH3 Fc domain of the composition of the present invention is derived from human IgG. In an embodiment, the hinge-CH2-CH3 Fc domain of the composition of the present invention is derived from human IgG1, IgG2 or IgG4.

[0107] In an embodiment, the linker of the composition of the present invention comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6. In an embodiment, the linker of the composition of the present invention 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 with SEQ ID NO: 6. In an embodiment, the linker of the composition of the present invention comprises an amino acid sequence having or 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).

[0108] In various aspects, the VHH domain of the composition of the invention is located at the N-terminus of the fusion or chimeric protein.

[0109] In various aspects, the recombinant GM-CSF of the composition of the invention is located at the N-terminus of the fusion or chimeric protein.

[0110] In an embodiment, the recombinant GM-CSF of the composition of the invention comprising the N-terminus of the fusion or chimeric protein produces increased yield as compared to a composition comprising recombinant GM-CSF at the C-terminus.

[0111] Functional properties of recombinant GM-CSF containing fusion or chimeric proteins

[0112] In 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., one or more functional parameters differ by no more than about 50%, or no more than about 40%, or no more than about 30%, or no more than about 20%, or no more than about 10%, or no more than about 5%, or no more than about 5-fold, or no more than about 4-fold, or no more than about 3-fold, or no more than about 2-fold of the functional parameter analyzed). In embodiments, the functional parameters of GM-CSF can be detected by assays known in the art, such as, but not limited to, proliferation assays using cells (such as the 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 (e.g., myeloid cell survival assays), cell differentiation assays, and co-culture experiments.

[0113] In embodiments, the fusion or chimeric proteins of the invention optionally having substitutions and / or deletions of the invention bind and / or activate granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R-α or CSF2R). In embodiments, the recombinant human GM-CSF molecules of the invention optionally having substitutions and / or deletions of the invention bind and / or activate granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R-α or CSF2R) with comparable affinity, efficacy and / or biological activity to wild-type human GM-CSF and / or sargramostim (e.g., one or more functional parameters differ by no more than about 50%, or no more than about 40%, or no more than about 30%, or no more than about 20%, or no more than about 10%, or no more than about 5%, or no more than about 5-fold, or no more than about 4-fold, or no more than about 3-fold, or no more than about 2-fold). Assays for GM-CSF binding and activation are known in the art. Non-limiting examples of such assays include, for example, radioligand assays or non-radioligand 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 by Biacore analysis. Whole cell ligand binding assays and cell-free assay systems using soluble GM-CSF receptor alpha (sGMRa) can also be used. Some other types of assays that can be used include receptor binding, or saturation binding, or competitive binding assays using radioiodinated GM-CSF, and cell proliferation assays.

[0114] In embodiments, the fusion or chimeric protein of the invention can be analyzed using one or more cell-based activity bioassays, for example, using a GM-CSF-dependent human cell line proliferation assay, for example, using 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.

[0115] In an embodiment, the efficacy of the fusion or chimeric protein of the invention is measured using a bioassay employing TF-1 cells, which is a human erythroleukemia cell line that proliferates in response to GM-CSF. The details of such an assay are known in the art. For example, a reference standard, control, and test sample are serially diluted in assay medium in triplicate and added to three separate 96-well plates. The TF-1 cells in suspension are then added and the mixture is incubated at 37°C for about 69.5-72 hours. After the addition of a fluorescent dye (e.g., ALAMARBLUE), the plate is incubated at 37°C for about 6.6-8 hours. The proliferation of the TF-1 cells is then measured in a fluorescent microplate reader.

[0116] In embodiments, the fusion or chimeric protein of the invention has about the same specific activity as recombinant human GM-CSF lacking the mutation (eg, as assayed using a bioassay employing TF-1 cells).

[0117] In embodiments, the functionality of a fusion or chimeric protein of the invention produced in a mammalian cell, such as a Chinese hamster ovary (CHO) cell, can be comparable to that of recombinant human GM-CSF produced in a yeast cell (e.g., one or more functional parameters differ by no more than about 90%, or no more than about 80%, or no more than about 70%, or no more than about 60%, or no more than about 50%, or no more than about 40%, or no more than about 30%, or no more than about 20%, or no more than about 10%, or no more than about 5%, or no more than about 10-fold, or no more than about 9-fold, or no more than about 8-fold, or no more than about 7-fold, or no more than about 6-fold, or no more than about 5-fold, or no more than about 4-fold, or no more than about 3-fold, or no more than about 2-fold).

[0118] In embodiments, the fusion or chimeric protein of the invention has improved pharmacokinetics and pharmacodynamics as compared to sargramostim or WT GM-CSF. In embodiments, the fusion or chimeric protein of the invention has an increased half-life as compared to sargramostim or WT GM-CSF (e.g., one or more functional parameters differ by no more than about 50%, or no more than about 40%, or no more than about 30%, or no more than about 20%, or no more than about 10%, or no more than about 5%, or no more than about 5-fold, or no more than about 4-fold, or no more than about 3-fold, or no more than about 2-fold).

[0119] In embodiments, the GM-CSF-R-α to which binding and / or activation occurs is expressed on the surface of a cell. In embodiments, the cell is a hematopoietic progenitor cell. In embodiments, the hematopoietic progenitor cell is an immune cell. In embodiments, the hematopoietic progenitor cell is irradiated.

[0120] In embodiments, the immunogenicity of the fusion or chimeric proteins of the invention, optionally with substitutions and / or deletions of the invention, is comparable to wild-type human GM-CSF and / or sargramostim (e.g., one or more functional parameters differ by no more than about 50%, or no more than about 40%, or no more than about 30%, or no more than about 20%, or no more than about 10%, or no more than about 5%, or no more than about 5-fold, or no more than about 4-fold, or no more than about 3-fold, or no more than about 2-fold). In embodiments, immunogenicity is analyzed using methods known in the art. Non-limiting examples include detecting one or more anti-GM-CSF binding antibodies as assessed by, for example, screening assays such as direct or indirect or bridging ELISA, electrochemiluminescence, bead-based chemiluminescence assays, radioimmunoprecipitation assays, surface plasmon resonance and biolayer interferometry, and cell-based luciferase reporter neutralizing antibody assays.

[0121] In various aspects, the fusion or chimeric protein significantly increases the level, cell count and / or amount of neutrophils, monocytes, eosinophils, total WBC and platelets. In embodiments, the fusion or chimeric protein significantly increases the level, cell count and / or amount of platelets.

[0122] In embodiments, the cell recombinant fusion or chimeric protein is soluble.

[0123] Nucleic acids and host cells

[0124] In an embodiment, a nucleic acid molecule encoding a fusion or chimeric protein as described herein is provided. In an embodiment, the nucleic acid molecule has a codon-optimized sequence.

[0125] In embodiments, the host cell is a yeast, mammalian, bacterial, insect, algae, or plant cell.

[0126] In embodiments, the yeast cell is a non-methylotrophic yeast cell.In embodiments, the host cell is a Saccharomyces cerevisiae cell.

[0127] In an embodiment, a human host cell expressing a nucleic acid molecule described herein is provided.

[0128] In an embodiment, a non-human host cell expressing a nucleic acid molecule described herein is provided.

[0129] In an embodiment, a CHO cell expressing a nucleic acid molecule described herein is provided.

[0130] Pharmaceutical compositions and preparations

[0131] In an embodiment, a pharmaceutical composition is provided, comprising a fusion or chimeric protein described herein and a pharmaceutically acceptable excipient or carrier.

[0132] Any pharmaceutical composition described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally contain a suitable amount of a pharmaceutically acceptable excipient in order to provide a form for proper administration.

[0133] In various embodiments, pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, when applied to a subject, a pharmaceutically acceptable excipient is sterile. When any agent described herein is administered intravenously, water is a useful excipient. Physiological saline solutions and dextrose aqueous solutions and glycerol solutions can 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, any agent described herein may also contain a small amount of a wetting or emulsifying agent or a pH buffering agent. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro ed., 19th ed. 1995), which is incorporated herein by reference.

[0134] The present disclosure includes the described pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any pharmaceutical composition of the present invention (and / or additional therapeutic agent) described herein can be in the form of a solution, suspension, emulsion, drops, tablets, pills, aggregates, capsules, capsules containing liquids, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen 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 another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In another embodiment, the pharmaceutical composition is formulated in a liquid.

[0135] If necessary, pharmaceutical compositions of the present invention (and / or additional therapeutic agents) may also include a solubilizing agent. In addition, the agent may be delivered with 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.

[0136] The formulations disclosed herein containing the pharmaceutical compositions of the present invention (and / or additional therapeutic agents) can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical field. Such methods generally include the step of associating the therapeutic agent with a carrier, which constitutes one or more auxiliary components. Typically, the formulation is prepared by uniformly and closely associating the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, molding the product into the dosage form of the desired formulation (e.g., wet or dry granulation, powder mixing, etc., followed by tableting using conventional methods known in the art).

[0137] 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 mode of administration described herein.

[0138] Administration routes include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or surface. Administration may be topical or systemic. In an embodiment, administration is achieved orally. In another embodiment, administration is by parenteral injection. The mode of administration may be at the discretion of the practitioner and depends in part on the site of the medical condition. In most cases, administration results in the release of any agent described herein into the bloodstream.

[0139] In certain embodiments, the fusion or chimeric protein or pharmaceutical composition thereof (and / or the additional therapeutic agent) is administered by an intravenous route.

[0140] In one embodiment, the pharmaceutical composition described herein (and / or additional therapeutic agent) is formulated as a composition suitable for oral administration according to conventional procedures. Compositions for oral delivery 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 include one or more agents, such as sweeteners, such as fructose, aspartame or saccharin; flavoring agents, such as mint, wintergreen oil or cherry; coloring agents; and preservatives to provide pharmaceutically palatable preparations. In addition, in the case of tablet or pill form, the composition may be coated with a coating to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over an extended period of time. Selectively permeable membranes around any pharmaceutical composition described herein (and / or additional therapeutic agent) driven by an osmotic active agent are also suitable for oral administration. In these latter platforms, fluids from the environment surrounding the capsule are absorbed by the driving compound, which swells to displace the agent or agent composition through the pores. These delivery platforms can provide a substantially zero-order delivery profile, in contrast to the tapered profile of an immediate release formulation. Time-delay materials (such as glyceryl monostearate or glyceryl stearate) can also be useful. Oral compositions can include standard excipients, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipient is pharmaceutical grade. In addition to the active compound, the suspension can also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, etc., and mixtures thereof.

[0141] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending agents or dispersants known in the art. Formulation components suitable for parenteral administration include sterile diluents, such as water for injection, physiological saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetates, citrates or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose.

[0142] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and a suitable mixture thereof.

[0143] Any pharmaceutical composition of the invention described herein (and / or additional therapeutic agent) can be administered by controlled release or sustained release means or by delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. 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 can be used to provide controlled release or sustained release of one or more active ingredients using, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, permeable systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide desired release profiles in different proportions. Suitable controlled release 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 agents described herein. Therefore, the present disclosure provides single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, caplets, and caplets suitable for controlled release or sustained release.

[0144] 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 by light of an appropriate wavelength, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

[0145] In another embodiment, a controlled release system can be placed adjacent to the target area to be treated, thereby 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) can be used.

[0146] The pharmaceutical preparation is preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. In the case where the composition is lyophilized, filtration sterilization can be performed before or after lyophilization and reconstitution.

[0147] Pharmaceutically acceptable salts and excipients

[0148] The compositions described herein may have sufficiently basic functional groups that can react with inorganic or organic acids, or carboxyl groups that can react with inorganic or organic bases to form pharmaceutically acceptable salts. As is well known in the art, pharmaceutically acceptable acid addition salts are formed with pharmaceutically acceptable acids. Such salts include, for example, the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

[0149] Pharmaceutically acceptable salts include, by way of non-limiting example, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, pamoates, phenylacetates, trifluoroacetates, acrylates, chlorobenzoates, dinitrobenzoates, hydroxybenzoates, benzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, alpha-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, decanoate, octanoate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, phthalate, terephthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0150] The term "pharmaceutically acceptable salts" also refers to salts of the compositions 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 and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxy-substituted mono-, di- or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, di- or tri-(2-OH-lower alkylamines), such as mono-, di- or tri-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tri-(hydroxymethyl)methylamine; N,N-di-lower alkyl-N-(hydroxy-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.

[0151] In embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

[0152] How to use

[0153] On the one hand, a method for treating a patient or subject who is undergoing or has undergone cancer therapy or is undergoing or has undergone a bone marrow transplant or has been acutely exposed to a myelosuppressive dose of radiation (e.g., acute radiation syndrome hematopoietic syndrome (H-ARS)) with or without other injuries, such as burns, wounds, infections, bleeding, or blunt trauma (e.g., radiation compound injury (RCI)) is provided; the method comprises administering to the patient a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof. In an embodiment, the patient is treated by regulating the clonal expansion, survival, differentiation, and activation state of hematopoietic progenitor cells. In an embodiment, the patient is treated by regulating the myelomonocytic lineage, by promoting the proliferation of megakaryocytes and erythroid progenitor cells. In an embodiment, the patient is treated by regulating hematopoietic progenitor cells, by stimulating the survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells. In an embodiment, after a bone marrow transplant, the patient is treated by regulating hematopoietic progenitor cells, by stimulating the survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells.

[0154] On the one hand, a treatment method is provided, which comprises administering to a patient a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof, or contacting cells with an effective amount of a pharmaceutical composition described herein and administering a therapeutically effective amount of the cells, wherein the therapy: accelerates neutrophil recovery and / or reduces the incidence of infection after induction chemotherapy; mobilizes hematopoietic progenitor cells into the peripheral blood for collection and transplantation by leukapheresis; accelerates myeloid reconstitution after autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation; treats delayed neutrophil recovery or transplant failure after autologous or allogeneic bone marrow transplantation; and / or treats acute radiation syndrome hematopoietic syndrome (H-ARS); and / or treats radiation-complexed injury (RCI).

[0155] In one aspect, a method for treating viral infection is provided, comprising: administering an effective amount of a composition comprising the fusion or chimeric protein of the present invention or a pharmaceutical composition comprising the same to a patient in need thereof.

[0156] In an embodiment, the viral infection is an influenza infection, optionally selected from influenza A, B, C and D virus infections.

[0157] In an embodiment, the viral infection is a viral hemorrhagic fever caused by a group of viruses optionally selected from the group consisting of Arenavirus, Flavivirus, Filovirus, Hantavirus, Nairovirus, Phenuvirus.

[0158] In embodiments, the lung infection is pneumonic plague caused by infection with Yersinia pestis.

[0159] In embodiments, the viral infection is a coronavirus infection. In embodiments, the coronavirus is a beta coronavirus, 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 embodiments, the coronavirus is an alpha coronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

[0160] Coronaviruses are members of the Coronaviridae family, including the recently known betacoronaviruses and alphacoronavirus respiratory pathogens that invade humans. 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.

[0161] Coronaviruses invade cells via the "spike" surface glycoprotein, which is responsible for viral recognition of angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor on mammalian hosts that facilitates viral entry into host cells. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable batorigin. Nature 2020. Novel coronavirus infection 2019 (COVID-19) is caused by SARS-CoV-2.

[0162] 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 the cause of a recent outbreak that has been declared a major threat to public health and a global emergency. Phylogenetic analysis of the complete genome of SARS-CoV-2 showed that the virus is most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sabeivirus). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, February 3, 2020.

[0163] SARS-CoV-2 is an enveloped single-stranded RNA virus that encodes a "spike" protein (also called S protein), which is a surface glycoprotein that mediates binding to cell surface receptors; integral membrane proteins; envelope proteins and nucleocapsid proteins. The S protein, which comprises 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, for example, 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 found. See Daniel et al., Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation. Science, February 19, 2020, which is incorporated herein by reference in its entirety.

[0164] Phylogenetic analysis of the complete genome of SARS-CoV-2 (GenBank accession number: MN908947) showed that the virus is most closely related to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sabeivirus) (89.1% nucleotide similarity). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, February 3, 2020, which is incorporated herein by reference in its entirety.

[0165] SARS-CoV-2 has a spike surface glycoprotein, a membrane glycoprotein M, an envelope protein E, and a nucleocapsid phosphoprotein N. The complete genome of the SARS-CoV-2 coronavirus (29903 nucleotides, single-stranded RNA) is described 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).

[0166] In embodiments, the method prevents or reduces the development of acute respiratory distress syndrome (ARDS) in the patient.

[0167] In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the patient has COVID-19. In embodiments, the patient has one or more of fever, cough, shortness of breath, diarrhea, upper respiratory tract symptoms, lower respiratory tract symptoms, pneumonia, and acute respiratory syndrome.

[0168] In the embodiment, the patient is hypoxic. In the embodiment, the patient suffers from respiratory distress. In the embodiment, the method improves the patient's oxygenation. In the embodiment, the method prevents or alleviates the patient's transition from respiratory distress to cytokine imbalance. In the embodiment, the method reverses or prevents cytokine storm. In the embodiment, the method reverses or prevents pulmonary or systemic cytokine storm. In the embodiment, the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome and hemophagocytic lymphohistiocytosis. In the embodiment, the method reverses or prevents the overproduction of one or more inflammatory cytokines. In the embodiment, inflammatory cytokines are IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10 and CCL7. One or more.

[0169] In embodiments, the method results in a decrease in the viral or bacterial load in the patient relative to before treatment.

[0170] In embodiments, the viral infection is selected from betacoronavirus infection, optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome-coronavirus (MERS-CoV), HCoV-HKU1 and HCoV-OC43 infection. In embodiments, the viral infection is selected from alphacoronavirus infection, optionally selected from HCoV-NL63 and HCoV-229E infection.

[0171] In embodiments, the betacoronavirus infection is severe acute respiratory syndrome (SARS).

[0172] In embodiments, the betacoronavirus infection is or is associated with coronavirus disease 2019 (COVID-19).

[0173] In embodiments, the viral infection is an influenza infection, optionally selected from influenza virus infection type A, type B, type C, and type D. In embodiments, the influenza infection is 2009 pandemic influenza A (H1N1) or avian influenza A (H5N1).

[0174] In an embodiment, the viral infection is a viral hemorrhagic fever caused by a group of viruses optionally selected from the group consisting of Arenavirus, Flavivirus, Filovirus, Hantavirus, Nairovirus, Phenuvirus.

[0175] In an embodiment, the lung infection is pneumonic plague caused by infection with Yersinia pestis.

[0176] In one aspect, a method for treating symptoms caused by exposure to chemical warfare agents is provided, the method comprising: administering to a patient in need thereof an effective amount of a composition comprising the fusion or chimeric protein of the present invention or a pharmaceutical composition comprising the same.

[0177] In embodiments, the chemical warfare agent is sulfur mustard (SM / HD) and / or chlorine gas.

[0178] In embodiments, the patient's exposure symptoms are characterized by myelosuppression.

[0179] In embodiments, provided methods result in reduction and / or improvement of symptoms resulting from exposure to sulfur mustard (SM / HD) and / or chlorine gas, including myelosuppression.

[0180] In one aspect, a method for treating and / or improving symptoms caused by a neurodegenerative disease in a patient or subject is provided; the method comprises administering to the patient a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof.

[0181] In an embodiment, 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.

[0182] In embodiments, the patient's neurodegenerative disease is characterized by oxidative stress, loss of neurite integrity, apoptosis, neuronal loss, or / and inflammatory response. In embodiments, the neurodegenerative disease is associated with cognitive impairment.

[0183] In one aspect, a method of treatment is provided, the method comprising administering a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof to a patient suffering from a neurodegenerative disease, or contacting cells with an effective amount of a pharmaceutical composition described herein and administering a therapeutically effective amount of the cells, wherein the therapy: induces a disease-modifying response; temporarily or permanently slows cognitive decline; results in symptom improvement; and / or slows the onset and / or progression of the disease.

[0184] In one aspect, the fusion or chimeric protein of the invention is provided as a composition that facilitates enhanced delivery of recombinant GM-CSF across the endothelial capillaries (blood-brain barrier, BBB) for central nervous system (CNS) therapeutics.

[0185] In one aspect, a method for treating and / or improving symptoms caused by an autoimmune disease in a patient or subject is provided; the method comprises administering to the patient a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof.

[0186] In one embodiment, the autoimmune disease is optionally selected from Crohn's disease and / or ulcerative colitis (UC) and / or irritable bowel disease (IBD).

[0187] In one aspect, a therapeutic method is provided, which comprises administering a therapeutically effective amount of a fusion or chimeric protein of the present invention or a pharmaceutical composition thereof to a patient suffering from an autoimmune disease, or contacting cells with an effective amount of a pharmaceutical composition described herein and administering a therapeutically effective amount of the cells, wherein the therapy can restore the balance between effector and regulatory immune function.

[0188] In one aspect, a method of treating and / or ameliorating symptoms caused by a caspase recruitment domain family member 9 (CARD9) deficiency in a patient or subject is provided.

[0189] In one aspect, a method of treating a patient or subject who is undergoing or has undergone therapy for a fungal infection is provided; the method comprises administering to the patient a therapeutically effective amount of a fusion or chimeric protein of the invention or a pharmaceutical composition thereof.

[0190] In embodiments, the compositions of the invention are used as an adjunct therapy to antifungal therapy.In embodiments, the compositions of the invention reduce and / or decrease the fungal load in various tissues.

[0191] In one aspect, a method of treating a patient or subject who is undergoing or has undergone therapy for hematopoietic syndrome with acute radiation syndrome (H-ARS) is provided.

[0192] In one aspect, a method of treating a patient or subject who is undergoing or has undergone therapy for radiation composite injury (RCI) is provided.

[0193] In one aspect, a method of treating a patient or subject who is undergoing or has undergone neurotomy therapy is provided.

[0194] In one aspect, a method of treating a patient or subject who is undergoing or has undergone therapy for a traumatic brain injury is provided.

[0195] In embodiments, the compositions of the invention are used as an adjunct therapy to antifungal therapy.In embodiments, the compositions of the invention reduce and / or decrease the fungal load in various tissues.

[0196] In one aspect, a method of treating a patient or subject who is undergoing or has undergone therapy for sepsis is provided. In embodiments, the compositions of the invention balance and / or modulate inflammation and acute phase responses and reduce morbidity and mortality.

[0197] In one aspect, a method of treating a patient or subject who is being or has been treated for multiple organ dysfunction syndrome (MODS) is provided.

[0198] In one aspect, a method of treating a patient or subject who is undergoing or has been treated for peripheral artery disease (PAD) is provided.

[0199] In one aspect, a method of treating a patient or subject who is undergoing or has undergone treatment for chronic wound healing is provided.

[0200] In one embodiment, the chronic wound is an optionally selected indolent wound of leprosy, leg ulcers and / or various other causes.

[0201] In an embodiment, the present composition is used as an adjunct therapy for wound healing therapies, including but not limited to hyperbaric oxygen therapy, ultrasound and electromagnetic therapy, and / or negative pressure wound therapy. In an embodiment, the present composition acts on various epidermal cells and enhances wound healing. In an embodiment, the present composition promotes tissue remodeling and regeneration.

[0202] Combination therapy and additional therapeutic agents

[0203] In various embodiments, the pharmaceutical compositions of the present disclosure are co-administered in combination with additional agents. Co-administration can be simultaneous or sequential.

[0204] In one embodiment, the additional therapeutic agent and the fusion or chimeric protein of the present disclosure or its pharmaceutical composition are administered to the subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the fusion or chimeric protein or its pharmaceutical composition are administered at a time interval of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. The administration of the additional therapeutic agent and the fusion or chimeric protein or its pharmaceutical composition can be performed by simultaneously administering a single formulation (e.g., a formulation comprising the additional therapeutic agent and the fusion or chimeric protein or its pharmaceutical composition) or a separate formulation (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the fusion or chimeric protein).

[0205] If their timing of administration makes the pharmacological activity of the additional therapeutic agent and the fusion or chimeric protein or its pharmaceutical composition overlap in time, so as to exert a combined therapeutic effect, then co-administration does not require simultaneous administration of therapeutic agents. For example, the additional therapeutic agent and the targeting portion (the fusion or chimeric protein or its pharmaceutical composition) composition can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the fusion or chimeric protein are administered at a time interval of more than about 60 minutes. For example, the time between the sequential administration of the additional therapeutic agent and the fusion or chimeric protein or its pharmaceutical composition may be separated by 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 one month. The optimal administration time will depend on the metabolic rate, excretion rate and / or pharmacodynamic activity of the additional therapeutic agent being administered and the fusion or chimeric protein or its pharmaceutical composition. The additional therapeutic agent or the fusion or chimeric protein composition or pharmaceutical composition thereof may be administered first.

[0206] Co-administration also does not require that the therapeutic agents be administered to the subject by the same route of administration. Instead, each therapeutic agent can be administered by any appropriate route (e.g., parenteral or non-parenteral).

[0207] In an embodiment, the fusion or chimeric protein described herein or its pharmaceutical composition acts synergistically when co-administered with another therapeutic agent. In such embodiments, the targeting moiety (composition of the present invention) and the additional therapeutic agent can be administered at a dose lower than the dose adopted when the agent is used in the context of a monotherapy.

[0208] In an embodiment, the additional therapeutic agent is an antiviral drug.

[0209] In an embodiment, the additional therapeutic agent is selected from the group consisting of antiviral agents such as remdesivir, favipiravir, oseltamivir, baloxavir, galisvir, amprenavir, tipranavir, saquinavir, nelfinavir, indinavir, darunavir, atazanavir, emetine, lopinavir and / or ritonavir, arbidol and lopinavir / ritonavir and / or ribavirin, darunavir and cobicistat and / or IFN-β-1b, β-D-N4-hydroxycytidine (NHC) (such as EIDD-1931 or EIDD-2801 or EIDD-2801); immunomodulators such as glucocorticoids, IFN-a 2a, IFN-a 2b, IFN-b, pegylated IFN-g, baricitinib, sirolimus, clazakizumab, canakinumab, XPro1595, tocilizumab, sarilumab, sildenafil, adalimumab, eculizumab, ivermectin, anakinra, prezcobix, xifentanyl, fingolimod, methylprednisolone, lelizumab, thalidomide, MK-2206, nicolasamide, nitazoxanide (nitazoxamide), chloroquine or hydroxychloroquine; antibiotics such as carlimycin, brelastatin, azithromycin, valinomycin, angiotensin inhibitors / antagonists (such as rhACE2 / GSK2586881 / APN01), losartan, eprosartan, telmisartan, valsartan; serine protease inhibitors, including camostat mesylate, nafamostat; other drugs such as bromhexine, aprotinin, chlorpromazine, zotatifin, methotrexate, lenalidomide, anti-VEGF-A and intravenous immunoglobulin (IVIG). For example, in embodiments, any of these additional therapeutic agents can be used in the case of SARS-CoV-2 infection.

[0210] In an embodiment, the additional therapeutic agent is selected from favipiravir, laninamivir octanoate, peramivir, zanamivir, oseltamivir phosphate, baloxavir marboxil, mifenovir, urumin, amantadine hydrochloride, rimantadine hydrochloride, adapromine, LASAG / BAY81-87981, celecoxib, etanercept, metformin, gemcitabine, dapivirine, trametinib, lisinopril, naproxen, nalidixic acid, dorzolamide, ruxolitinib, midodrine, diltiazem; statins, including atorvastatin, nitazoxanide; PPAR antagonists, including gemfibrozil. For example, in an embodiment, any of these additional therapeutic agents can be used in the case of influenza infection or other viral infection (such as those causing viral hemorrhagic fever).

[0211] In an embodiment, the additional therapeutic agent is a drug that treats a neurodegenerative disease.

[0212] In an embodiment, the additional therapeutic agent is selected from levodopa, cholinesterase inhibitors (including donepezil (ARICEPT), rivastigmine (EXELON), galantamine (RAZADYNE)), atypical antipsychotics / second generation antipsychotics (including serotonin-dopamine antagonists (SDA), pleiotropic receptor targeted antipsychotics (MARTA) and D 2 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-nerve growth factor), and deep brain stimulation. For example, in embodiments, any of these therapeutic agents can be used in the context of neurodegenerative diseases.

[0213] In embodiments, the additional therapeutic agent is an antifungal drug.

[0214] In embodiments, the additional therapeutic agent is selected from nystatin; amphotericin B; terbinafine; a medical device; a pyrimidine analog, such as flucytosine; a triazole, such as fluconazole, isavuconazole, itraconazole, praconazole, ravuconazole, voriconazole, and posaconazole; an imidazole, such as clotrimazole, econazole, ketoconazole, and miconazole; an echinocandin, such as caspofungin, micafungin, and anidulafungin. For example, in embodiments, any of these therapeutic agents may be used in the setting of fungal infection.

[0215] In an embodiment, the additional therapeutic agent is a drug to treat an autoimmune disease.

[0216] In an embodiment, the additional therapeutic agent is selected from corticosteroids, such as prednisone and budesonide (Entocort EC); oral 5-aminosalicylate, such as sulfasalazine (AZULFIDINE); anti-inflammatory drugs / inflammatory inhibitors / immunosuppressants, such as azathioprine (AZASAN, IMURAN), mercaptopurine (PURINETHOL, PURIXAN), methotrexate (TREXALL), and corticosteroids and aminosalicylate, such as mesalamine (ASACOL HD, DELZICOL), balsalazide (COLAZAL), tofacitinib (XELJANZ), cyclosporine (GENGRAF, NEORAL, SANDIMMUNE), and olsalazine (DIPENTUM); biologics such as natalizumab (TYSABRI), vedolizumab (ENTYVIO), infliximab (REMICADE), adalimumab (HUMIRA), cecilizumab (CIMZIA), ustekinumab (STELARA), and golimumab (SIMPONI); antibiotics such as ciprofloxacin (CIPRO) and metronidazole (FLAGYL); antidiarrheal drugs such as loperamide (IMODIUM AD); analgesics; vitamins and supplements, including iron supplements. For example, in embodiments, any of these therapeutic agents can be used in the context of an autoimmune disease.

[0217] In an embodiment, the additional therapeutic agent is a drug that accelerates or aids wound healing or other dermatological disorders.

[0218] In an embodiment, the additional therapeutic agent is selected from 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 calcipotriol 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; Gokman therapy; anthralin; light therapy, such as natural sunlight, UVB broadband , narrow-band UVB, psoralen plus ultraviolet A (PUVA), and excimer laser therapy; immunosuppressants such as steroids, methotrexate (TREXALL), adalimumab (HUMIRA), infliximab (REMICADE), and cyclosporine; the biologics etanercept (ENBREL), infliximab (REMICADE), adalimumab (HUMIRA), ustekinumab (STELARA), secukinumab (COSENTYX), and ethelezumab (TALTZ); thioguanine (TABLOID) and hydroxyurea (DROXIA, HYDREA), aloe extract cream, fish oil supplements, Oregon grape, and essential oils.

[0219] In embodiments, the additional therapeutic agent is a cardiovascular disease or peripheral arterial disease drug.

[0220] In an embodiment, the additional therapeutic agent is selected from an anticoagulant, such as apixaban (ELIQUIS), warfarin (COUMADIN); antiplatelet and dual antiplatelet therapy (DAPT), such as aspirin together with a P2Y12 inhibitor, such as clopidogrel (PLAVIX), prasugrel (EFFIENT), ticagrelor (BRILINTA), dipyridamole (PERSANTINE); 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 (β-adrenergic blockers), such as acebutolol (SECTRAL); combined alpha and beta blockers, such as carvedilol (COREG, COREG CR); calcium channel blockers, such as amlodipine (NORVASC); cholesterol-lowering drugs, such as statins (atorvastatin / LIPITOR), niacin (nicotinic acid / niacin); cholesterol absorption inhibitors (ezetimibe / ZETIA), combined statins and cholesterol absorption inhibitors (ezetimibe / simvastatin (Vytorin); digitalis (digoxin / LANOXIN); diuretics, such as acetazolamide (DIAMOX); vasodilators, such as isosorbide dinitrate (ISORDIL) and nitroglycerin (NITRO BID, NITROSTAT).

[0221] In embodiments, the additional therapeutic agent is a drug for treating sepsis or septicemia.

[0222] In an embodiment, the additional therapeutic agent is selected from an antibiotic, such as ceftriaxone (ROCEPHIN), cefotaxime (CLAFORAN), ampicillin and sulbactam (UNASYN), ceftazidime (FORTAZ), and clindamycin (CLEOCIN).

[0223] In an embodiment, the additional therapeutic agent is a drug for treating an oncological indication.

[0224] In an embodiment, the additional therapeutic agent is selected from a cytotoxic agent, including toxins such as ricin; a chemotherapeutic agent such as 5-fluorouracil, a radioisotope (e.g., iodine-131, radium-223), a targeted therapy (e.g., HERCEPTIN), and an immuno-oncology agent (e.g., ipilimumab (YERVOY) and nivolumab (OPDIVO)).

[0225] In embodiments, the additional therapy is a drug that increases platelet cell number or count and / or treats immune thrombocytopenia (ITP).

[0226] In an embodiment, the additional therapeutic agent is selected from a steroid such as prednisone, immunoglobulin therapy, romiplostim (NPLATE), eltrombap (PROMACTA), and rituximab (RITUXAN, TRUXIMA).

[0227] Preparation method

[0228] In various aspects, a method for preparing a fusion or chimeric protein is provided, the method comprising: (a) obtaining cells transfected with a nucleic acid encoding recombinant human GM-CSF or an extract thereof, a linker, and a humanized single domain antibody, the nucleic acid comprising an amino acid sequence having at least about 97% identity to SEQ ID NO: 1 or SEQ ID NO: 2 and optionally having a substitution or deletion at position N37 or its corresponding position; (b) purifying the fusion or chimeric protein from the transfected cells using one or more HPLC columns; and (c) collecting a purified fusion or chimeric protein, wherein the purified chimeric protein is substantially free of a highly mannosylated form of GM-CSF.

[0229] In embodiments, the cell is a prokaryotic or eukaryotic host cell, such as a yeast, mammalian, bacterial, insect, algae, or plant cell.

[0230] Suitable prokaryotic host cells include bacterial cells (eg, Escherichia coli, Bacillus subtilis, Mycobacterium, Mycobacterium tuberculosis, or other appropriate bacterial cells) and archaeal cells (eg, Methanococcus jannaschii and Methanococcus maritima).

[0231] In an embodiment, 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 (eg, mammalian cells, avian cells, and fish cells), and plant cells.

[0232] Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells.

[0233] Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Imperfectomycetes.

[0234] Suitable yeast host cells include, but are not limited to, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, and Yarrowia. In embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Kluyveromyces, Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0235] Suitable filamentous fungal host cells include, for example, any filamentous form of the subdivision Eumycota and Oomycetes. In an embodiment, the filamentous fungal host cell can be a cell of the following species: Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypo crea, Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae and the genus Aspergillus niger. In one embodiment, the filamentous fungus is Aspergillus niger.

[0236] In various aspects, a method for preparing a composition comprising a fusion or chimeric protein is provided, the method comprising: (a) obtaining cells transfected with a nucleic acid encoding recombinant human GM-CSF or an extract thereof, a linker and a VHH humanized Nanobody, the nucleic acid 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 its corresponding position, (b) purifying the fusion or chimeric protein from the transfected cells using one or more HPLC columns, (c) collecting the purified fusion or chimeric protein, the purified fusion or chimeric protein being substantially free of a highly mannosylated form of GM-CSF.

[0237] In embodiments, the cell is a yeast cell, such as, but not limited to, Saccharomyces cerevisiae.

[0238] In embodiments, the cell is a mammalian cell, such as, but not limited to, a Chinese Hamster Ovary (CHO) cell.

[0239] In embodiments, production of the fusion or chimeric protein in mammalian cells increases expression levels of the compositions of the invention as compared to production using non-mammalian cells.

[0240] In embodiments, the method further comprises formulating the purified fusion or chimeric protein for injection, such as subcutaneous or intravenous injection.

[0241] sequence

[0242] SEQ ID NO: 1 is wild-type GM-CSF. The sites of substitution or deletion of some embodiments are indicated by underlining and bold:

[0243]

[0244]

[0245] SEQ ID NO: 2 is sargramostim. The sites of substitution or deletion of some embodiments are indicated by underlining and bolding:

[0246]

[0247] SEQ ID NO: 3 is the human VH3-23 sequence.

[0248] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSC AASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK.

[0249] SEQ ID NO: 4 is the G4S linker sequence.

[0250]

[0251] SEQ ID NO:5 is the G4A linker sequence.

[0252]

[0253] SEQ ID NO:6 is a variant of the hinge sequence from the human IgG1 sequence:

[0254] EPKSSDKTHTSPPSPAPE.

[0255] SEQ ID NO: 7 is the sequence of the VHH-GMCSF construct, wherein the anti-HSA VHH is located at the N-terminus, GM-CSF is located at the C-terminus, and G4S serves as a linker.

[0256]

[0257]

[0258] SEQ ID NO: 8 is the sequence of the VHH-GMCSF construct, in which the anti-HSA VHH is located at the N-terminus, GM-CSF is located at the C-terminus, and the hinge serves as a linker.

[0259]

[0260] SEQ ID NO: 9 is the sequence of the VHH-GMCSF construct, in which GM-CSF is located at the N-terminus, anti-HSA VHH is located at the C-terminus, and the hinge serves as a linker.

[0261]

[0262] SEQ ID NO: 10 is the sequence of the VHH-GMCSF construct, wherein GM-CSF is located at the N-terminus, anti-HSA VHH is located at the C-terminus, and G4S serves as a linker.

[0263]

[0264]

[0265] SEQ ID NO: 11 is the sequence of the VHH-GMCSF construct, wherein the anti-HSA VHH is located at the N-terminus, GM-CSF is located at the C-terminus, and G4A serves as a linker.

[0266]

[0267] SEQ ID NOs: 12-14 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).

[0268] SEQ ID NO:15-17 are CDRs: CDR-H1:ETLDYYAIG (SEQ ID NO:15), CDR-H2:IASSGGSTNYADSVKG (SEQ ID NO:16), CDR-H3:AVLECRTVVRGYDY (SEQ ID NO:17).

[0269] definition

[0270] The following definitions are used in conjunction with the invention disclosed herein. Unless defined otherwise, 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.

[0271] When used in combination with an agent effective for treating coronavirus infection, an "effective amount" is an amount effective for treating or ameliorating coronavirus infection.

[0272] As used herein, "a / an" or "the" may mean one or more than one. In addition, when used in conjunction with a reference number indication, the term "about" means the reference number indication plus or minus up to 10% of the reference number indication. For example, the language "about 50" covers a range of 45 to 55.

[0273] As mentioned herein, unless otherwise specified, all composition percentages are by weight of the total composition. As used herein, the wording "including" and its variations are intended to be non-restrictive, so that the items stated in the list do not exclude other similar items that may also be applicable to the materials, compositions, devices and methods of this technology. Similarly, the terms "can" and "may" and their variations are intended to be non-restrictive, so that the statement that an embodiment can or may include certain elements or features does not exclude other embodiments of the present invention technology that do not contain those elements or features.

[0274] Although the open-ended term "comprising" is used herein as a synonym for terms such as including, containing, or having, alternative terms such as "consisting of" or "consisting essentially of" may alternatively be used to describe the invention or embodiments thereof.

[0275] The present invention is further illustrated by the following non-limiting examples.

[0276] Example

[0277] Example 1: Discovery and humanization of VHH molecules

[0278] Two camels were immunized with human serum albumin (HSA) to produce antibodies. Total RNA was isolated from the cells followed by cDNA synthesis. Using this cDNA, the heavy chain variable domain of the camelid heavy chain only antibody (VHH) coding sequence was amplified by PCR. The structure of the heavy chain IgG (hcIgG) is shown in Figure 1A Shown in.

[0279] Subsequently, fluorescence activated cell sorting (FAC) combined with fluorescently labeled HSA was used to isolate single VHHs that bind to HSA. Figure 1B These VHHs also do not bind albumin at an epitope that would block FcRn binding, which is important for the recycling and pharmacokinetic (pK) properties of albumin. The studies also showed that the clones do not bind non-specifically to membrane preparations. Finally, there was no change in the binding properties of the VHH clones when measured between pH 5.5 and 7.4. After the HSA-binding VHH clones were identified, the VHHs were humanized by transplanting the CDRs into appropriate human VHHs and using appropriate back mutations and camelid-specific sequences required to maintain VHH stability.

[0280] The three anti-HSA VHH clones identified and humanized were used for experimental fusion proteins to demonstrate that VHHs contribute to increased serum half-life. Protein fusion molecules were prepared to study the potential half-life extension ability of the three VHHs. Fab molecule fusion proteins were constructed with each of the three VHHs. Murine models demonstrated that VHHs contribute to the half-life extension of Fab fragments, indicating that the half-life of Fab increased by 17 times. VHH59-1 also extended the half-life of other anti-HSA-VHH molecules by more than double ( Figure 1C ).

[0281] Example 2: Identification and construction of GMCSF-VHH

[0282] Four variants were initially prepared and characterized. The orientation of VHH and GMCSF varied around two linker sequences: VHH-linker-GMCSF or GMCSF-linker-VHH. Either (GGGGS)x4 or (GGGGA)x4 linkers were used. The second sequence was a modified hinge sequence of human IgG1, as the VHH is directly linked to the hinge region of camel's IgG, i.e., the connection between the VHH and the constant region of the camel's natural antibody. The albumin leader sequence was chosen instead of the native GMCSF leader sequence because data showed that the cleavage of the leader sequence was heterogeneous when evaluated using SignalP software analysis, while the albumin leader sequence was uniformly cleaved. The 4 constructs were cloned and transiently expressed in CHO cells. The secretory leader sequence was identical for all 4 constructs. The sequence was in Figure 2 Shown in.

[0283] Example 3: Characterization of VHH-GA-GMCSF* molecules

[0284] Multiple variants of anti-HSA-VHH59-1 were prepared, allowing us to empirically determine which variant had the best developability characteristics, including expression titer, aggregation propensity as determined by size exclusion chromatography, ability of the VHH to bind HSA in the context of a fusion or chimeric protein, biological activity of the fused GMCSF, and binding characteristics of GMCSF to its cognate receptor. The *GM-CSF sequence used for the fusion was identical to the GMCSF variant sequence previously used to express CHO-derived GM-CSF variants.

[0285] The expression titer levels of the four constructs in transient CHO are shown in Table 1. The expression level of GM-CSF at the N-terminus was much higher than that at the C-terminus. A 30% loss in yield was observed due to relatively weak binding between the VHH and the Pro A resin. However, this GMCSF-hinge-VHH also appeared to contain more aggregates compared to the other constructs.

[0286] Table 1:

[0287]

[0288] The ability of the VHH portion of the fusion or chimeric proteins to bind to human serum albumin (HSA) was assessed using a ForteBio-based assay. Interestingly, all four VHH-GMCSF fusion or chimeric proteins were able to bind HSA only when they were immobilized on the sensor and the albumin was in solution, but not when the albumin was mobile on the sensor.

[0289] In the biological activity assay, TF-1 cells were used in the presence of ( Figure 3A ) and the absence of albumin ( Figure 3B) was evaluated for the ability of GM-CSF within the fusion or chimeric protein to bind to the cognate receptor. In response to exposure to the fusion or chimeric GM-CSF, TF-1 cells proliferated. The four chimeric or fusion proteins were compared to constructs containing a CHO-derived GMCSF* variant (VHH-GMCSF) as well as LEUKINE and commercial GM-CSF. The data indicate that VHH-G4Sx4-GM-CSF and GM-CSF-G4Sx4-VHH are biologically active in the TF-1 cell-based assay, i.e., cells proliferate when the GM-CSF fusion or chimeric protein is added to the culture medium. However, it was noted that the GMCSF-G4Sx4-VHH EC50 (Table 2) was slightly lower, or less active, than the other orientations. The fusion or chimeric proteins containing CHO-derived GM-CSF* showed similar activity to LEUKINE. Thus, the results show that the orientation of GM-CSF relative to VHH within the fusion or chimeric protein has an impact on its biological activity in the presence and absence of albumin in a cell-based assay, and that inhibition of GM-CSF activity is greater in the presence of albumin. Without wishing to be bound by theory, albumin, when bound to VHH, may result in a change in the ability of GM-CSF (at the N-terminus) to interact with its receptor, and thus albumin is unable to induce proliferation of TF1 cells as compared to GM-CSF alone, or when GM-CSF is fused to the C-terminus of the VHH-linker ( Figure 3A -B).

[0290] Table 2 :

[0291] EC50 Leukine 0.003041 CHO-GMCSF* 0.00299 VHH-G4A-GMCSF* 0.009436 Buffer 0

[0292] Example 4: Mass spectrometric evaluation of glycan profiles of fusion proteins

[0293] Mass spectrometry was used to verify the N-terminal cleavage heterogeneity and glycosylation of the molecule. Mass spectrometry identified that the (G4Sx4) linker had xylose added, which is known to be immunogenic to humans. See Arch Biochem Biophys. 426: 266-278. Mass spectrometry also showed that the known glycosylation sites Ser7, Ser10 and N27 in GM-CSF were also glycosylated throughout the VHH-G4Sx4-GM-CSF variant and were similar to the GMCSF* produced by CHO used in the fusion protein. Six different stable lines were analyzed and the results are listed below. High-resolution liquid chromatography-mass spectrometry (LC-MS) (Vanquish UPLC Q Ex active, Thermo) was used to determine the complete molecular weight and glycan profile ( Figure 4A). This figure shows the glycan analysis by mass spectrometry, in particular the glycan occupancy of the 6 stable CHO clones that make up VHH-G4A GMCSF*. 100 μg of sample was diluted to 2 mg / mL with deionized water and then injected into LC-MS. The data were analyzed and deconvoluted by BioPharmaFinder software to obtain the intact protein molecular mass. Glycosylation species were identified by accurate mass shift and manual calculation. Based on the intensity of all identified ions, assuming that their ionization efficiency is comparable, the N / O glycosylation occupancy was estimated (Table 3).

[0294] Table 3 :

[0295]

[0296] Constructs generated from these six clones were analyzed in a TF-1 cell based assay. The data showed that the activity did not change even when the N-linkage occupancy at N27 was varied between about 12% and about 53%. Thus, the activity of the molecule did not change and did not appear to be affected by the presence or absence of glycans ( Figure 4B and Table 4).

[0297] Table 4 :

[0298] clone EC50 10B7 0.00479 9F6 0.00509 6G7 0.00504 10A8 0.00506 2E7 0.00612 6E7 0.00523

[0299] Example 5: In vitro binding characterization of VHH-GMCSF constructs

[0300] To evaluate binding to albumin, VHHs were cloned onto human IgG1 Fc and hinge for ease of purification and analysis. The binding affinity of the VHH-Fc constructs was determined by BLI using Octet 384 (Pall Life Sciences). The VH-Fc constructs were loaded onto the AHC sensor and then exposed to monomeric targets (human, mouse, or rhesus monkey albumin) in solution for sequential association and dissociation in buffer solution (Table 5 and Table 6). Figure 5A -C).

[0301] Table 5 :

[0302]

[0303]

[0304] To determine the dissociation constant, binding reactions and dissociations were performed at the described pH. Data acquisition was performed at 30°C.

[0305] The graph shows response units on the Y-axis (binding of target to VHH-Fc on the chip sensor) and time on the X-axis ( Fig. 6A -D). Data (time and response units) were analyzed using ForteBio data analysis software. Data were fitted to a 1:1 binding model to calculate association (Kassoc) and dissociation rates (Kdissociation), and KD was calculated using the ratio kd / ka. Table 6 shows the measured and calculated parameters for each target and each pH.

[0306] Table 6 :

[0307]

[0308] Example 6: In vivo characterization of VHH-GA-GMCSF*

[0309] In a monkey study (one monkey per molecule tested), VHH-GA-GMC SF* and two control molecules (CHO produced GMCSF* and Leukine) were experimentally tested. After IV injection of 20ug VHH-GA-GM-CSF* or 10ug Leukine or CHO produced GM-CSF*, the pK of each of the three molecules and the biological effects on circulating hematopoietic cells were measured over a period of time. Since the molecular weight of VHH-GA-GM-CSF* is approximately twice that of GM-CSF*, VHH-GA-GM-CSF* was administered at a higher concentration, and by doubling the amount, equimolar amounts of the molecules were administered. Blood draws were performed at 12min, 30min, 1, 2, 4, 8, 24, 48, and 72 hours after injection. Data ( Fig. 7A -C) shows that the VHH in the VHH-GA-GMCSF* construct contributes to a significant increase in pK from 0.79 hours to >10 hours. Leukine and CHO-produced GMCSF* showed similar half-lives (t 1 / 2 ), indicating that CHO-produced GMCSF is not responsible for the half-life extension. A single rhesus monkey was injected intravenously with 10 ug / Kg of the listed forms of GMCSF. Blood was drawn at 12 min, 30 min, 1, 2, 4, 8, 24, 48 and 72 hours after injection. The blood was analyzed for cell content and the construct concentration in the blood was determined by construct-specific proprietary ELISA. VHH-GA-GMCSF* and VHH-GA-GMCSF* show approximately 17-fold higher t than the Leukine or CHO-produced GMCSF forms used in the VHH-GA-GMCSF constructs. 1 / 2 .

[0310] After blood was drawn from the injected monkeys, complete hematopoiesis was assessed using FACS analysis using cell-specific markers for cell count and cell type ( Fig. 8A-E). The numbers of neutrophils, monocytes, eosinophils, total WBCs, and platelets were determined ( Fig. 8A -E). Both CHO-produced GM-CSF* and LEUKINE showed similar dramatic increases in neutrophil, monocyte and WBC cell counts 24 hours after injection, returning to baseline by day 4. However, fusion or chimeric VHH-GA-GMCSF* neutrophil, monocyte and WBC cell counts peaked on day 2 and returned to baseline between days 8-12. The longer duration of neutrophil, monocyte and WBC counts after injection of the fusion or chimeric protein indicates Fig. 7A -C results in a longer duration biological readout of increased blood cells in the circulation. Similar delayed kinetics of increased eosinophils were observed following injection of the fusion or chimeric protein. The number of neutrophils, monocytes, eosinophils, and WBCs also increased following injection of the chimeric or fusion protein. Surprisingly, the data show that platelet numbers increased significantly following injection of the fusion or chimeric VHH-GA-GMCSF* as compared to CHO produced GM-CSF* and LEUKINE.

[0311] Equivalent solutions

[0312] 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. Such equivalents are intended to be encompassed within the scope of the appended claims.

[0313] Incorporated by Reference

[0314] All patents and publications cited herein are hereby incorporated by reference in their entirety.

[0315] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally 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. The composition of claim 1, wherein the recombinant human GM-CSF protein comprises an amino acid sequence that is at least about 97% identical 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 having 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 its counterpart is a polar and neutrally charged hydrophilic amino acid.

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

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

7. The composition of claim 6, wherein the polar and 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. A composition as described in claim 8 or 9, wherein the VHH comprises an amino acid sequence that is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical to SEQ ID NO: 3, and / or comprises three CDRs selected from (i) SEQ ID NO: 12-14 or variants thereof or (ii) SEQ ID NO: 15-17 or variants thereof.

11. The composition of any one of claims 8-10, wherein the VHH further comprises a substitution or deletion within the framework region relative to the 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 comprises substantially glycine and serine residues.

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 composition of any one of claims 13-15, wherein the flexible linker comprises (Gly4Ser) 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. The composition of claim 18, wherein the hinge-CH2-CH3 Fc domain is derived from human IgG1, IgG2 or IgG4.

20. The composition of claim 12, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, 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: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. The composition of claim 20, wherein the linker comprises an amino acid sequence that is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical to SEQ ID NO:

6.

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

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

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

11.

25. The composition of any one of claims 1-24, wherein the composition binds to and / or activates 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. The composition of claim 27, wherein the hematopoietic progenitor cells are immune cells.

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

30. The composition of any one of claims 1-29, wherein the composition binds human, mouse, and rhesus monkey serum albumin.

31. The composition of claim 30, wherein the composition binds 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 HSA for binding to the neonatal Fc receptor (FcRn).

34. The composition of any of the preceding claims, wherein the fusion or chimeric protein is soluble.

35. The composition of any of the preceding claims, wherein the fusion or chimeric protein exhibits substantially similar function to sargramostim.

36. The composition of any of the preceding claims, wherein the recombinant human GM-CSF comprises multiple molecular forms.

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

38. The composition of any one of the preceding claims, wherein the recombinant human GM-CSF is substantially free of high mannosylated forms.

39. A nucleic acid molecule encoding a fusion or chimeric protein or a fragment thereof as claimed in any one of the preceding claims.

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

41. A human host cell expressing the nucleic acid molecule of claim 39 or 40.

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

43. A non-human host cell expressing the nucleic acid molecule of claim 39 or 40.

44. The non-human host cell of claim 43, wherein the host cell is a yeast cell.

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

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

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

48. A method for treating a patient or subject who is undergoing or has undergone cancer therapy or is undergoing or has undergone a bone marrow transplant or has been acutely exposed to myelosuppressive doses of radiation (e.g., H-ARS) and / or suffers from radiation complex injury (RCI); the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as described in claim 47.

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

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

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

53. A method of therapy, comprising Administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 47, or contacting cells with an effective amount of the pharmaceutical composition of claim 47 and administering a therapeutically effective amount of the cells, The therapies described: accelerate neutrophil recovery and / or reduce the incidence of infection after induction chemotherapy; mobilization of hematopoietic progenitor cells into peripheral blood for collection and transplantation by leukapheresis; Accelerate myeloid reconstitution after autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation; Treatment of delayed neutrophil recovery or graft failure following autologous or allogeneic bone marrow transplantation; and / or Treatment of hematopoietic acute radiation syndrome (H-ARS); Treating radiation composite injury (RCI); Treating neurodegenerative diseases; Treat nerve severance; Treating traumatic brain injuries; Treat lung infections; Treating an autoimmune disease, optionally Crohn's disease or IBD; Treating sepsis / septicemia; Treating invasive fungal infections; Treat multiple organ dysfunction syndrome (MODS); Treatment of peripheral artery disease (peripheral artery disease / peripheral arterial disease) / cardiovascular disease; Tumor treatment indications Aids in wound healing and / or treatment of dermatological conditions.

54. A method for treating a coronavirus infection, the method comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

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

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

58. The method of claim 56, wherein the patient has COVID-19.

59. A method for treating an influenza virus infection, the influenza virus optionally being selected from type A, type B, type C and type D, the method comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

60. A method of treating viral hemorrhagic fever caused by a virus optionally selected from the group consisting of Arenavirus, Flavivirus, Filovirus, Hantavirus, Nairovirus, Phenuvirus, comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

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

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

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

64. The method of any one of claims 54-63, wherein the patient suffers from respiratory distress.

65. The method of any one of claims 54-64, wherein the method prevents or reduces the development of acute respiratory distress syndrome (ARDS) in the patient.

66. The method of any one of claims 54-65, wherein the method improves oxygenation of the patient.

67. The method of any one of claims 54-66, wherein the method prevents or reduces the transition from respiratory distress to cytokine imbalance in the patient.

68. The method of any one of claims 54-67, wherein the method reverses or prevents a cytokine storm.

69. The method of claim 68, wherein the method reverses or prevents pulmonary or systemic cytokine storm.

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. The method of claim 70, wherein the method reverses or prevents overproduction of one or more inflammatory cytokines.

72. The method of claim 71, wherein 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.

73. The method of any one of claims 54-72, wherein the method results in a reduction in the viral or bacterial load in the patient relative to before treatment.

74. A method of treating and / or ameliorating symptoms resulting from exposure to a chemical warfare agent, the method comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

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

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

77. The method of any one of claims 74-76, wherein the composition causes improvement in symptoms of chemical exposure including myelosuppression.

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

79. The method of claim 78, wherein the neurodegenerative disease is selected from the group consisting of 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.

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

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

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

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

84. The method of any one of claims 78-83, wherein the composition causes improvement in symptoms of a neurodegenerative disease.

85. The method of any one of claims 78-84, wherein the composition slows the onset and / or progression of the neurodegenerative disease.

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

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

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

89. The method of claim 88, wherein the method treats an invasive fungal infection.

90. The method of claim 89, wherein the invasive fungal infection is selected from the group consisting of candidiasis, mucormycosis, invasive aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, frog dung mold, otomycetoma, cryptococcal meningitis, and histoplasmosis.

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

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

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

94. The method of any one of claims 88-93, wherein the composition helps reduce and / or decrease fungal load in various tissues.

95. A method of treating and / or assisting wound healing and / or dermatological conditions, the method comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

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

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

98. A method of treating cancer, the method comprising: An effective amount of the pharmaceutical composition of claim 47 is administered to a patient in need thereof.

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

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 growth, survival, metastasis, epithelial-mesenchymal transition, immune escape or recurrence.

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

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

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

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

106. The composition of any one of claims 1-38, wherein the composition facilitates delivery of recombinant GM-CSF across the endothelial capillaries (blood-brain barrier, BBB) for central nervous system (CNS) therapeutics.

107. The composition of any one of claims 1-38, wherein the fusion or chimeric protein is produced by transfecting mammalian cells, optionally CHO cells, wherein the fusion or chimeric protein exhibits increased half-life as compared to sargramostim.

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

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

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

111. The composition of any one of claims 1-38, wherein the fusion or chimeric protein significantly increases the level, cell count and / or amount of platelets.

112. The composition of any one of claims 1-38, wherein the fusion or chimeric protein is produced by transfecting mammalian cells, optionally CHO cells, wherein the chimeric protein exhibits improved pharmacodynamics as compared to sargramostim.

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

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