CN116507355A

CN116507355A - Granulocyte macrophage colony stimulating factor mutant

Info

Publication number: CN116507355A
Application number: CN202180072791.7A
Authority: CN
Inventors: M·费尔德豪斯; J·约斯特
Original assignee: Partner Therapy Ltd
Current assignee: Partner Therapy Ltd
Priority date: 2020-10-26
Filing date: 2021-10-25
Publication date: 2023-07-28

Abstract

The present invention is based on the finding that amino acid substitutions in the sequence of a saxitin result in a product with simplified manufacturability.

Description

Granulocyte macrophage colony stimulating factor mutant

Cross Reference to Related Applications

The present application claims the benefit and priority of U.S. provisional patent application Ser. Nos. 63/105,425 and 63/177,481, filed on 26 10 and 21, 2021, which are hereby incorporated by reference in their entireties.

Technical Field

The present invention relates generally to compositions and methods relating to mutant granulocyte-macrophage colony-stimulating factor (GM-CSF).

Sequence listing

The present application contains a sequence listing, which is submitted via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2021, 10 months and 20 days, named "PNR-003PC_ST25.Txt" and was 4,096 bytes in size.

Background

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

GM-CSF is a blood growth factor that regulates the production, migration, proliferation, differentiation and function of hematopoietic cells. In response to inflammatory stimuli, GM-CSF is released by various cell types including T lymphocytes, macrophages, fibroblasts, and endothelial cells. GM-CSF then activates and enhances the production and survival of neutrophils, eosinophils and macrophages. Natural GM-CSF is typically produced near its site of action in regulating proliferation, differentiation and survival of hematopoietic progenitor cells in vitro, but only at picomolar concentrations (10 ^-10 To 10 ^-12 M) is present in circulating blood. See A lexander 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.

Human GM-CSF (hGM-CSF) was synthesized as a precursor protein of 144 amino acid residues with a 17 amino acid signal peptide. The method comprisesThe precursor protein was processed to produce a mature protein of 127 amino acids with a predicted molecular weight of 14.4kDa. It has two disulfide linkages that migrate as broad bands of 15-30kDa due to glycosylation and sialylation. N-linked glycans at Asn ²⁷ And Asn ³⁷ At this point, and multiple potential sites for O-linked glycosylation exist at Ser5, ser7, ser9 and Ser10, the range of glycan structures at these sites has not been well defined. 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 1991.265,4483-4491; zhang Q et al Proc.Natl.Acad.Sci.2014,2885-2890.

Recombinant human granulocyte-macrophage colony stimulating factor (rhu GM-CSF) has been FDA approved for use in combination with chemotherapy in the treatment of neutropenia, hematological malignancies, and malignancies such as leukemia. Clinically, GM-CSF for use in the treatment of post-chemotherapy neutropenia and aplastic anemia 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 documented. See Dorr RT. Clin therapeutics 1993.15 (1): 19-29; armitage JO.blood 1998,92:4491-4508; kovacic JC et al J Mol Cell cardiol.2007,42:19-33; jacobs PP et al Microbial Cell Factories 2010,9:93.

Despite the existence of five classes of heterologous protein production platforms, including bacteria, yeast, plants, insect cells, and mammalian cells, more than 50% of the biopharmaceuticals currently commercially available are produced in mammalian cell lines. This is due in part 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.

The GM-CSF expression system affects the pharmacokinetic properties, biological activity, and clinical toxicity of GM-CSF. Clinically, GM-CSF has been produced in chinese hamster ovary cells (CHO-GM, rella stim), escherichia coli (escherichia coli-GM, molgrastim) or yeast (yeast-GM, sargrastim). See Dorr RT. Clin therapeutics 1993.15 (1): 19-29; walsh G.Nat Biotechnol.2006,24:769-776; jacobs PP et al Nat Protoc.2009,4:58-70; jacobs PP et al Microbial Cell Factories 2010,9:93; walsh G.Nat Biotechnol.2018,36 (12): 1136-1145. During the manufacture of GM-CSF, there are four main GM-CSF isoforms/classes: "hyperglycosylated" (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 only via recombinant technology using yeast (saxitin) or Chinese Hamster Ovary (CHO) cell (rapamycin) technology that produces complex glycoform mixtures, but not using escherichia coli. Glycan heterogeneity reflects the lack of specificity in CHO cell post-translational glycosylation. See Zhang Q et al PNAS.111 (8): 2885-2890.

The expression vector of the Shagrastim contains leader sequences directing transcription in https:// en.wikipedia. Org/wiki/rough_endoplasmic_reticum Rough endoplasmic reticulum and secretion by mature GM-CSF cells. N-glycosylation occurs when glycans are added to the nitrogen of asparagine (Asn) or arginine (Arg) side chains, and O-glycosylation occurs when glycans are added to the oxyhydroxy side chains of serine (Ser) or threonine (Thr) or tyrosine (Tyr) amino acids. Asparagine residues typically require a consensus sequence of Asn-Xxx-Ser/Thr/Cys, where Xxx may be any amino acid other than proline for N-glycosylation. The rhu GM-CSF protein sequence of the sagrastim contains two consensus sequences, asn-Leu-SerNLS) And Asn-Glu-ThrNET) Wherein the protein may be potentially glycosylated. During the manufacture of the sagrastim, two N-glycosylation sites Asn ²⁷ And Asn ³⁷ Is glycosylated in some GM-CSF species present in the fermentor. Thr (Thr) ³⁹ (or Ser ³⁹ Or Cys ³⁹ ) The residues being such that Asn ²⁷ Or Asn ³⁷ Important anchor points where glycosylation is possible. The class of N-oligosaccharides attached at asparagine residue 37 is referred to as the "hyperglycosylated" class (30-100 kDa) because these glycoforms contain up to hundreds of mannose residues. Only high mannosylation occurs at N37. Previous reports have been explored Mutagenesis that may affect glycosylation (e.g., gene 55:287-293 (1987) and Hehring Inst. Mitt.83:1-7 (1988)), however, these studies focused on protein yields and did not evaluate activity or attempted to streamline the purification process (see, e.g., table 2 of Hehring Inst. Mitt.83:1-7 (1988)).

In the upstream manufacturing process, four major GM-CSF species, "hyperglycosylated" (about 50 kDa), N-and N- +O-glycosylated (about 20kDa, peak 2), O-glycosylated (about 16kDa, peak 3) and non-glycosylated (about 15kDa, peak 4) species are present in the partially purified fermentation broth. The "hyperglycosylated" GM-CSF species is considered to be non-product and is removed in downstream processes, resulting in a product, namely the drug substance (BDS) containing the three major glycoforms (peaks 2-4). A small amount of oxidized GM-CSF (.ltoreq.4%) was detected in BDS and DP, which was labeled "Peak 1" in the T-0075 inverted glycoform assay. See Walter P et al JCB.1981.91 (2 Pt 1): 545-50; cantrell MA et al PNAS.1985.82:6250-6254. Most proteins synthesized in the https:// en.wikipedia. Org/wiki/rough_endoplasmic_reticum Rough endoplasmic reticulum undergo glycosylation, which is an enzyme-directed site-specific process. See Medzihradszky KF. Methods Mol biol.2008.446:293-316.Varki A et al, essential of glycobiology, chapter 56, 3 rd edition.

In addition to the three major rhu GM-CSF glycoforms currently present in the formulation, there are less than 6% of the "hyperglycosylated" forms of the product. However, this "hyperglycosylated" form is not present in current drug products of the saxitin (LEUKINE). It is estimated that the isolated "hyperglycosylated" species contains two N-acetylglucosamine residues plus an average of 43 mannose residues, which are at the low end of the range known to exist in fermentation broths. The class of N-oligosaccharides attached at asparagine residue 37 is referred to as the "hyperglycosylated" class (30-100 kDa) because these glycoforms contain up to hundreds of mannose residues. See Strenler KE et al 1994. This "hyperglycosylated" species was completely removed during downstream purification and was not detected in the final saxifrage drug substance (BDS) used to make the saxifrage.

The final product of the sagrastim consists of N-glycosylated and O-glycosylated forms as well as non-glycosylated forms. Thus, the sauce-pavilion has heterogeneity in its glycoform profile, which is always very consistent throughout its license history. The three major glycoforms in the saxitin can be separated on an SDS-PAGE gel. The historical average relative amounts of these three glycoforms were quite consistent, 28.68% ± 0.86%, 22.58% ± 0.73% and 48.74% ± 0.8%, respectively. Approximately half of the GM-CSF protein in the sagrastim 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 a significant portion of the molecule, including the H1 and H5 alpha-helices, without impeding the receptor binding site and some H6 alpha-helices and the C-terminal random coil.

Although the carbohydrate component of recombinant GM-CSF varies based on the cell source, receptor binding or in vitro and in vivo biological activity does not require glycosylation. The yeasts rhu GM-CSF and the non-glycosylated E.coli-derived rhu GM-CSF have comparable specific activities measured by both receptor affinity and cell proliferation specific activity. See Urdial and Park,1988; metcalf D.cancer 1990:65:2185-2195.

Endogenous proteins in humans degrade at different rates in the body, which may vary greatly due to their function. For example, hemoglobin persists throughout the life cycle of erythrocytes, and histones have half-lives measured in years, while other proteins such as ornithine decarboxylase (11 minutes half-life) have very fast degradation rates. See Thomas E Creig hton (1993), "Chapter 10-Degradation". Proteins: structures and Molecular Properties (2 nd 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 intracellular homeostasis in the processing and degradation of proteins, including those proteins that regulate cell cycle progression, apoptosis, and DNA repair. See Heinemeeyer W et al J Biol chem 1997.272:25200-25209. Lysosomes also remove proteins that are not needed in the cytoplasm and outside the cell. Substances from outside the cell are taken up by endocytosis, whereas substances from inside the cell are digested by autophagy. See Settembre C et al Nature Reviews Molecular Cell biology.2013.14 (5): 283-96.GM-CSF has been shown to have a half-life that can be extended from 50 minutes to 85 minutes. 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 is largely dependent on their bioavailability. See Dorr rt.clinical Therapeutics/volume 15, stage 1, 1993.

Bioavailability of therapeutic proteins (biotherapeutic or biological) in humans can also be affected by the route of administration of the drug. See Dorr RT, clinical Therapeutics/volume 15, stage 1, 1993. Almost all therapeutic proteins are immediately available after Intravenous (IV) administration, but then may be degraded over time by proteases present in the blood plasma. In some cases, subcutaneous (SC) administration prolongs exposure to proteins with short elimination half-lives by maintaining high plasma concentrations for longer periods of time, and is better tolerated than intravenous administration. Thus, the absorption of a biotherapeutic drug following subcutaneous 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 the sauce-station into the systemic circulation may be advantageous for different pathways, because of their different molecular sizes. Two non-glycosylated rhuGM-CSF Leu23 species in Shagrastim (approximately 14kDa on SDS PAGE gels, consisting of full-length molecules and truncated Ala ³ des-A ¹ P ² The amino acid sequences of the species determine mw= 14,430.47 and 14266.31) should obviously be favourable for diffusion into capillaries and thus the bioavailability of these protein molecules in the blood stream should be very fast. Approximately eight O-glycosylated glycoforms containing 1-8 mannose glycoresidues (approximately 16kDa on SDS PAGE gels, with MW between 14,500 and 16,000 as determined by mass spectrometry) may be delivered to the systemic circulation through lymphatic capillaries and capillaries. However, contains two n-acetylglucosamine residues and N-and N- +O-glycosylated glycoforms of 6-27 mannose residues (approximately 21kDa on SDS PAGE gels, MW between 16,200 and 19,000 as determined by mass spectrometry) may strongly favor diffusion and absorption by the lymphatic pathway in view of their molecular size.

The GM-CSF expression system can affect pharmacokinetic parameters in which the degree of glycosylation affects GM-CSF half-life, distribution, and elimination. The relationship between the degree of GM-CSF glycosylation and its distribution, clearance and activity was reported using a rat model. The specific activity of GM-CSF, measured in vitro, was found to be significantly reduced in the largest, most fully glycosylated form of the protein relative to smaller, less glycosylated molecules. The effective half-life of GM-CSF in rat blood stream after a single intravenous bolus injection 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 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.

The "hyperglycosylated" rhu GM-CSF species have been shown to have a statistically significant lower biological activity, whereas the Specific Activities (SA) of the 19.5, 16.8 and 15.5kDa species are very similar. The SAs of the other three glycoforms N-and N- +O-glycosylated (21 kDa, peak 2), O-glycosylated (16 kDa, peak 3) and non-glycosylated species (14 kDa, peak 4) have been shown to have no statistical differences from the values obtained for the parent BDS s-sargrastim at 99% confidence levels.

There remains a need for GM-CSF molecules suitable for use in simple purification methods and which are substantially free of "hyperglycosylated" species.

Disclosure of Invention

Thus, the present invention relates to mutant forms of GM-CSF that provide, inter alia, unique glycoform profiles relative to wild-type or sabcometin.

In aspects, a recombinant human GM-CSF protein is provided comprising 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, E38 and/or T39 or corresponding thereto, e.g., the amino acid at position N37 or corresponding thereto may be substituted for polar and charge neutral hydrophilic amino acids such as glutamine (Q), serine (S), threonine (T), proline (P) and cysteine (C); the amino acid at position E38 or at the position corresponding thereto may be substituted with a hydrophobic aliphatic amino acid such as alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V), and/or is not proline; and/or the amino acid at position T39 or a position corresponding thereto may be substituted with alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M) and valine (V).

In an embodiment, the recombinant human GM-CSF of the invention is functionally similar to a sargrastim. In embodiments, the recombinant human GM-CSF of the invention comprises a plurality of molecular forms, such as non-glycosylated, O-glycosylated, N-glycosylated, and/or N+O-glycosylated forms. In embodiments, the recombinant human GM-CSF of the invention is substantially free of hyperglycosylated forms, e.g., high mannose glycosylated forms. In an embodiment, recombinant human GM-CSF of the invention resolves into three peaks when quantified by reverse phase high performance liquid chromatography (RP-HPLC). In embodiments, recombinant human GM-CSF of the invention does not provide a significant peak at a retention time of less than about 20 minutes when quantified by reverse phase high performance liquid chromatography (RP-HPLC). In embodiments, recombinant human GM-CSF of the invention is substantially free of hyperglycosylated forms, such as hypermannosylated forms, when purified without the use of organic solvents (e.g., without limitation, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid, and/or n-propanol) and/or reverse phase C4 HPLC columns for the purpose of purification and/or removal of hyperglycosylated peaks.

In embodiments, recombinant human GM-CSF produced in mammalian cells is functionally enhanced compared to recombinant human GM-CSF produced in yeast cells.

In embodiments, the production titer of recombinant human GM-CSF produced in mammalian cells is increased compared to that produced in yeast cells.

In embodiments, nucleic acid molecules encoding recombinant human GM-CSF (e.g., codon optimized sequences) of the invention are also provided. In an embodiment, a non-human host cell (e.g., a yeast cell, such as a non-methylotrophic yeast cell, e.g., saccharomyces cerevisiae) expressing a nucleic acid molecule encoding a recombinant human GM-CSF of the present invention is also provided. In an embodiment, there is also provided a pharmaceutical composition comprising recombinant human GM-CSF of the invention and a pharmaceutically acceptable excipient or carrier.

In some aspects, a method of treating a patient or subject undergoing or having undergone cancer therapy or undergoing or having undergone bone marrow transplantation and/or having been acutely exposed to a myelosuppressive dose of radiation is provided; the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition described herein.

In aspects, there is provided a method of treating a viral infection, such as, but not limited to, a coronavirus infection, such as, but not limited to, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering an effective amount of a pharmaceutical composition described herein, or a method of treating or preventing a viral infection in a subject in need thereof by providing plasma from a donor subject that has recovered from a viral infection, such as, but not limited to, a coronavirus infection, such as, but not limited to, SARS-CoV-2, the plasma comprising IgG, igM, and/or IgA antibodies to the virus causing the infection, and the donor subject has been treated with a recombinant human GM-CSF protein described herein to stimulate the production of the antibodies; and administering the plasma to a subject in need thereof.

In an aspect, there is provided a method of preparing a recombinant producing a composition comprising recombinant human GM-CSF, the method comprising: (a) Obtaining a yeast cell or extract thereof transfected with a nucleic acid encoding a recombinant human GM-CSF comprising an amino acid sequence having at least about 97% identity to SEQ ID No. 2 and having a substitution or deletion at positions N37, G38 and/or T39 or corresponding thereto; (b) Purifying GM-CSF from the transfected yeast cells using one or more HPLC columns, wherein purification is performed in the absence of an organic solvent (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 purification and/or removal of hyperglycosylated peaks; and (c) collecting purified GM-CSF that is substantially free of hyperglycosylated, e.g., high mannose, forms of GM-CSF.

Drawings

FIG. 1 shows Saccharomyces expression of GM-CSF as quantified by HPLC. Asterisks identify high mannose glycosylated GM-CSF peaks.

FIG. 2 shows Western blotting for confirming protein identity.

FIG. 3 shows a reversed phase HPLC assay for determining the percentage distribution of rhu GM-CSF glycoform.

FIG. 4 shows a comparative chromatogram of BDS LEUKINE (reference standard) versus CHO expression of rhu GM-CSF grown in yeast. BDS LEUKINE refers to wild type GM-CSF, SEQ ID NO:1, having NO T39A or N37Q amino acid substitution within its sequence.

FIG. 5 shows a comparative size exclusion chromatogram of BDS LEUKINE (reference standard) versus CHO expression of rhu GM-CSF grown in yeast.

FIG. 6 shows the fermentation titers of rhu GM-CSF grown in yeast versus CHO cells.

FIG. 7 shows a comparative functional TF-1 bioassay measuring the biological activity of rhu GM-CSF with mutations T39A and N37Q, as well as LEUKINE (i.e., GM-CSF without T39A or N37Q).

FIG. 8 shows TF-1 assay data for measuring the bioactivity of rhu GM-CSF with mutation N37Q grown in CHO cells, compared to 3 different batches of BDS LEUKINE (Leukine BDS new material stored at 2℃to 8℃for 12 months and Leukine at-70℃for 48 months) stored using different conditions.

Detailed Description

The present invention is based in part on the following findings: single amino acid changes in the sequence of a sauce-station (e.g., at positions 37, 38 and/or 39 of a sauce-station or equivalent) result in functional GM-CSF that does not require purification with an organic solvent (e.g., 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 the hyperglycosylated peaks observed in the case of a sauce-station.

Compositions of GM-CSF

In one aspect, an engineered GM-CSF protein is provided.

In embodiments, the engineered GM-CSF useful in the practice of the invention includes any pharmaceutically safe and effective GM-CSF, or any derivative thereof having the biological activity of GM-CSF and the substitutions and/or deletions of the invention.

In one embodiment, the engineered GM-CSF used to carry out the subject methods is derived from recombinant human GM-CSF (rhu GM-CSF), such as a sauce pavilion (LEUKINE). The sargrastim is a biosynthetic, yeast-derived recombinant human GM-CSF that has a single 127 amino acid glycoprotein with leucine instead of proline at position 23, which differs from endogenous human GM-CSF. Other natural and synthetic GM-CSF and derivatives thereof having the biological activity of natural human GM-CSF are equally useful in the practice of the invention.

In an embodiment, the recombinant human GM-CSF molecule of the invention is glycosylated. In embodiments, the recombinant human GM-CSF molecules of the invention comprise one or more substitutions and/or deletions that affect glycosylation of GM-CSF.

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, R.T., clin. Ther.15:19-29,1993; horgaard et al, eur. J. Hematol.50:32-36,1993).

In one aspect, 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 a substitution or deletion at position N37, E38 and/or T39 or positions corresponding thereto.

In embodiments, the amino acid at position N37 or corresponding to position of recombinant human GM-CSF is a polar and charge neutral hydrophilic amino acid. In some embodiments, the polar and charge neutral hydrophilic amino acids are selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the polar and charge neutral hydrophilic amino acid is glutamine (Q).

In embodiments, the amino acid at position E38 or corresponding to position E38 of recombinant human GM-CSF is a hydrophobic aliphatic amino acid and/or is not proline. In embodiments, the hydrophobic aliphatic amino acid is selected from the group consisting of alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In embodiments, the amino acid at position T39 or corresponding to position T39 of recombinant human GM-CSF is a hydrophobic aliphatic amino acid. In embodiments, the hydrophobic aliphatic amino acid is selected from the group consisting of alanine (a), glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V). In embodiments, the hydrophobic aliphatic amino acid is alanine (a). In embodiments, the amino acid at position T39 or at the position corresponding thereto of recombinant human GM-CSF is uncharged. In embodiments, the amino acid at position T39 or corresponding to position T39 of recombinant human GM-CSF is not glutamic acid (E).

In embodiments, 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 an N37 deletion, an N37Q, N37 82348 37T, N37P, N37C, E38 deletion, an E38A, E38L, E38I, E8238 8235 deletion, a T39A, T39G, T39L, T39 6239M and a T39V or corresponding mutation.

In embodiments, recombinant human GM-CSF has the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, or a variant thereof having substitution and/or deletion of the present invention having about 90%, or about 93%, or about 95%, or about 97%, or about 98% identity thereto.

In an embodiment, GM-CSF is one of moraxella, sarcandat, and rapaxella with substitutions and/or deletions of the present invention.

Without wishing to be bound by theory, the core of hGM-CSF consists of four angularly arranged helices. The crystal structure of rhGM-CSF and mutagenesis analysis (Rozwater ski D A et al, proteins26:304-13, 1996) showed that, in addition to the nonpolar side chains in the protein core, 10 buried hydrogen bonding residues involved intramolecular hydrogen bonding to the backbone atoms which were more conserved than residues hydrogen bonding 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 was located in the gap between the secondary structural elements. The two surface clusters of hydrophobic side chains are located near the intended receptor binding region. Mutagenesis of residues on the A/C-side of the helix confirmed the importance of certain Glu, gly and Gln residues. Thus, these residues will not be substituted in the functional substitution variants of hGM-CSF used in the present invention, and these helices will remain in the functional fragments or deletion variants of hGM-CSF used in the present invention.

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

The N-terminal helix of hGM-CSF controls high affinity binding to its receptor (Shanafelt A B et al, EMBO J10:4105-12,1991). Transduction of the biological effects of GM-CSF requires interaction with at least two cell surface receptor components, one of which is shared with the cytokine IL-5. The above studies identified receptor binding determinants in GM-CSF by locating unique receptor binding domains on a range of human-mouse hybrid GM-CSF cytokines. The interaction of GM-CSF with the shared subunit of its high affinity receptor complex is controlled by a small portion of the peptide chain. The presence of several key residues in the N-terminal alpha-helix of (a) is sufficient to confer interaction specificity.

Thus, in embodiments, this information may inform the skilled artisan about acceptable changes in amino acid sequence.

In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative substitutions and/or non-conservative substitutions.

"conservative substitutions" may be made, for example, based on the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into the following six standard amino acid groups: (1) hydrophobicity: met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr; asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

As used herein, a "conservative substitution" is defined as the exchange of an amino acid for another amino acid listed in the same set of six standard amino acid sets shown above. For example, asp is exchanged by Glu to retain a negative charge in the polypeptide so modified. In addition, glycine and proline may be substituted for each other based on their ability to disrupt the alpha-helix.

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

In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine β -alanine, GABA and δ -aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, α -aminoisobutyric acid, 4-aminobutyric acid, abu, 2-aminobutyric acid, γ -Abu, epsilon-Ahx, 6-aminocaproic acid, aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocysteine, sulfoalanine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β -alanine, fluoro-amino acids, designer amino acids such as β -methylaminoacid, cα -methylaminoacid, nα -methylaminoacid, and general amino acid analogs.

Modification of the amino acid sequence may be accomplished using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al Molecular Cloning: A Laboratory Manual, cold Spring Harbor Press, plainview, N.Y.,1989 and Ausubel et al Current Protoc ols in Molecular Biology, john Wiley & Sons, new York, N.Y.,1989.

Sugar type

In embodiments, the recombinant human GM-CSF molecules of the invention comprise a plurality of molecular forms. In embodiments, the molecular form is selected from the group consisting of non-glycosylated forms, O-glycosylated forms, N-glycosylated forms, and n+o glycosylated forms.

In embodiments, the recombinant human GM-CSF is substantially free of hyperglycosylated forms, e.g., high mannose glycosylated forms. In embodiments, the recombinant human GM-CSF has about or less than about 10% of the high mannose form after purification, or about or less than about 5% of the high mannose form after purification, or about or less than about 3% of the high mannose form after purification, or about or less than about 2% of the high mannose form after purification, or about or less than about 1% of the high mannose form after purification.

In embodiments, the recombinant human GM-CSF has less highly mannosylated form than wild-type human GM-CSF and/or a sargrastim when expressed and purified in the same manner. In embodiments, the recombinant human GM-CSF has about 100%, or about 90%, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20% or about 10% less high mannose-ylated form than wild-type human GM-CSF and/or sargrastim when expressed and purified in the same manner. In embodiments, the amount of the high mannose glycosylated form may 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., gel staining procedures based on a periodic acid-schiff (PAS) reaction), affinity-based methods such as using a sugar binding protein (e.g., lectin), 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 methods (eastern blot).

In an embodiment, the recombinant human GM-CSF of the invention is suitable for simpler purification than the purification used for the preparation of the saxitin.

In embodiments, the mutant GM-CSF proteins of the invention are resolved into three glycoforms and lack a hyperglycosylated form, e.g., by HPLC. Comparing FIG. 1, it shows four peaks in GM-CSF as resolved on HPLC-asterisks identify high mannose glycosylated GM-CSF peaks.

In embodiments, GM-CSF proteins of the invention can be purified without the need for hyperglycosylated 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 C4HPLC column for purification and/or removal of hyperglycosylated peaks. In embodiments, the GM-CSF protein of the invention can be purified without the need for hyperglycosylated species, without the need for acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid, and/or N-propanol. In embodiments, the GM-CSF protein of the invention may be purified without a reverse phase C4HPLC column for the purpose of purification and/or removal of hyperglycosylated peaks.

In an embodiment, recombinant human GM-CSF of the invention resolves into three peaks when quantified by reverse phase high performance liquid chromatography (RP-HPLC). In embodiments, recombinant human GM-CSF does not provide a significant peak at a retention time of less than about 20 minutes when quantified by reverse phase high performance liquid chromatography (RP-HPLC).

In embodiments, the recombinant human GM-CSF of the invention comprises more than one species (e.g., glycoforms). In embodiments, none of the species has a molecular weight greater than about 20 kDa.

In embodiments, recombinant human GM-CSF is substantially free of hypermannosylated forms when purified without the use of an organic solvent (e.g., without limitation, 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 hyperglycosylated peaks.

In embodiments, recombinant human GM-CSF produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, is a monomer having a heterogeneous glycoform, with glycosylated and non-glycosylated isoforms.

Functional Properties of recombinant GM-CSF

In embodiments, the recombinant human GM-CSF molecules of the invention having substitutions and/or deletions of the invention are functionally similar to wild-type human GM-CSF and/or to a sand-box (e.g., the 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-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 assayed). In embodiments, 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 TF-1 cell lines, primary bone marrow cells, biochemical assays such as ILITE (EAGLE) GM-CSF (luciferase under control of GM-CSF promoter), cell survival assays (e.g., bone marrow cell survival assays), cell differentiation assays, and co-culture experiments.

In an embodiment, the recombinant human GM-CSF molecule of the invention with the substitutions and/or deletions of the invention binds to and/or activates granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R- α or CSF 2R). In embodiments, recombinant human GM-CSF molecules of the invention having substitutions and/or deletions of the invention bind and/or activate granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R- α or CSF 2R) with comparable affinity, efficacy, and/or bioactivity as wild-type human GM-CSF and/or sagrastim (e.g., 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-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(s). 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 blotting, 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.

In embodiments, one or more cell-based activity bioassays may be used to determine the recombinant human GM-CSF molecules of the invention, e.g., using a GM-CSF dependent human cell line proliferation assay, e.g., 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.

In an embodiment, the potency of the recombinant human GM-CSF molecules of the invention is measured using a bioassay employing TF-1 cells, which are human erythroleukemia cell lines that proliferate in response to GM-CSF. The details of this assay are known in the art. For example, reference standards, controls, and test samples are serially diluted in triplicate in assay medium and added to three separate 96-well plates. TF-1 cells in suspension were then added and the mixture incubated at 37℃for 69.5-72 hours. After addition of the fluorescent dye (e.g., ALAMARBLUE), the plates were incubated at 37 ℃ for 6.6-8 hours. TF-1 cell proliferation was then measured in a fluorescent plate reader.

In embodiments, the recombinant human GM-CSF molecules of the invention have about the same specific activity as recombinant human GM-CSF lacking the mutation (e.g., as determined using a bioassay employing TF-1 cells).

In embodiments, recombinant human GM-CSF produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, is functionally enhanced compared to recombinant human GM-CSF produced in yeast cells.

In embodiments, GM-CSF-R- α is expressed on the surface of a cell that undergoes binding and/or activation. In embodiments, the cells are hematopoietic progenitor cells. In embodiments, the hematopoietic progenitor cells are immune cells. In embodiments, the hematopoietic progenitor cells are irradiated.

In embodiments, the recombinant human GM-CSF molecules of the invention having substitutions and/or deletions of the invention are comparable in immunogenicity to wild-type human GM-CSF and/or to a sargrastim (e.g., the 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-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 determined using methods known in the art. Non-limiting examples include detection of 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 chemiluminescent assays, radioimmunoprecipitation assays, surface plasmon resonance and biolayer interferometry, and cell-based luciferase reporter gene-neutralizing antibody assays.

In embodiments, the cellular recombinant human GM-CSF is soluble.

Nucleic acids and host cells

In embodiments, nucleic acid molecules encoding the recombinant human GM-CSF described herein are provided. In embodiments, the nucleic acid molecule has a codon optimized sequence.

In embodiments, non-human host cells expressing the nucleic acid molecules described herein are provided.

In embodiments, the host cell is a yeast, mammalian, bacterial, insect, algal, or plant cell.

In embodiments, the yeast cell is a non-methylotrophic yeast cell. In embodiments, the host cell is a saccharomyces cerevisiae cell.

In embodiments, CHO cells expressing the nucleic acid molecules described herein are provided.

Pharmaceutical composition and formulation

In an embodiment, a pharmaceutical composition comprising recombinant human GM-CSF described herein and a pharmaceutically acceptable excipient or carrier is provided.

Any of the pharmaceutical compositions 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 comprise a suitable amount of a pharmaceutically acceptable excipient in order to provide a form for proper administration.

In various embodiments, the pharmaceutical excipients may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical excipients may be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline and dextrose in water and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso r. Gennaro, 19 th edition 1995), which is incorporated herein by reference.

The invention includes the described pharmaceutical compositions (and/or additional therapeutic agents) in various formulations. Any of the pharmaceutical compositions (and/or additional therapeutic agents) of the invention described herein may take the form of a solution, suspension, emulsion, drop, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dry powder, or any other suitable form for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

The pharmaceutical compositions (and/or additional therapeutic agents) of the present invention may also contain a solubilizing agent, if desired. In addition, the agent may be delivered using suitable vehicles or delivery devices known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device.

The formulations of the invention comprising the pharmaceutical compositions of the invention (and/or additional therapeutic agents) may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of associating the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the formulation is prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into dosage forms (e.g., wet or dry granulation, powder blends, etc., and then tableting using conventional methods known in the art) of the desired formulation.

In various embodiments, any of the pharmaceutical compositions (and/or additional therapeutic agents) described herein are formulated according to conventional procedures into compositions suitable for the modes of administration described herein.

Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or topical. Administration may be local or systemic. In some embodiments, administration is achieved orally. In another embodiment, the administration is by parenteral injection. The mode of administration may be at the discretion of the practitioner and will depend in part on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the blood.

In particular embodiments, GM-CSF (and/or an additional therapeutic agent) is administered by intravenous route.

In one embodiment, the pharmaceutical compositions (and/or additional therapeutic agents) described herein are formulated according to conventional procedures as compositions suitable for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. The combination for oral administration may comprise one or more agents, for example a sweetener, such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, oil of wintergreen, or cherry red; a colorant; and a preservative to provide a pharmaceutically palatable preparation. Furthermore, in the case of a tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over an extended period of time. Selectively permeable membranes around any of the pharmaceutical compositions (and/or additional therapeutic agents) described herein driven by an osmotically active agent are also suitable for use in compositions for oral administration. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the driven compound, which swells to pass through the pore displacer or agent composition. These delivery platforms may provide a substantially zero order delivery profile (delivery profile), as opposed to a tapered profile of an immediate release formulation. Delay materials such as glyceryl monostearate or glyceryl stearate may also be useful. Oral formulations may include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. In one embodiment, the excipient is pharmaceutical grade. Suspensions, in addition to the active composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth and the like, and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) which may be dissolved or suspended in a sterile injectable medium immediately prior to use. They may contain, for example, suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and agents for modulating tonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiological saline; antibacterial water; cremophor ELTM (BASF, parsippany, N.J.) or Phosphate Buffered Saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be antimicrobial preserved. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

Any of the pharmaceutical compositions of the invention (and/or additional therapeutic agents) described herein may 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, 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 may be used to provide controlled or sustained release of one or more active ingredients using, for example, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide the desired release profile in varying proportions. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The present invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, caplets and caplets suitable for controlled or sustained release.

The controlled or sustained release of the active ingredient may be stimulated by different conditions including, but not limited to, a change in pH, a change in temperature, stimulation via light of an appropriate wavelength, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

In another embodiment, the controlled release system may be placed adjacent to the target area to be treated, thereby requiring only a portion of the systemic dose (see, e.g., goodson, medical Appli cations of Controlled Release, supra, volume 2, pages 115-138 (1984)). Other controlled release systems discussed in the review of Langer,1990,Science 249:1527-1533) may be used.

The pharmaceutical formulation is preferably sterile. Sterilization may be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization may be performed before or after lyophilization and reconstitution.

Pharmaceutically acceptable salts and excipients

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 from pharmaceutically acceptable acids. Such salts include, for example, those described in Journal of Pharmaceutical Science,66,2-19 (1977) and The Handbook of Pharmaceutical Salts; pharmaceutically acceptable salts listed in Properties, selection, and use.p.h.stahl and c.g.weruth (editorial), verlag, zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

Pharmaceutically acceptable salts include, as non-limiting examples, sulfate, citrate, acetate, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucarate, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate hydroxybenzoates, methoxybenzoates, methylbenzates, o-acetoxybenzoates, naphthalene-2-benzoates, isobutyrates, phenylbutyrates, alpha-hydroxybutyrates, butyne-1, 4-dicarboxylic acid salts, hexyne-1, 4-dicarboxylic acid salts, decanoates, octanoates, cinnamates, glycolates, hippurates, malates, hydroxymaleates, malonates, mandelates, methanesulfonates, nicotinates, phthalates, terephthalates, propiolates, propionates, phenylpropionates, sebacates, suberates, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethylsulfonates, 2-hydroxyethylsulfonates, methylsulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, naphthalene-1, 5-sulfonates, xylene sulfonate and tartrate.

The term "pharmaceutically acceptable salt" refers to salts of the compositions of the present invention having acidic functionalities such as carboxylic acid functionalities 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, dicyclohexylamines; 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 tris- (hydroxymethyl) methylamine; n, N-di-lower alkyl-N- (hydroxy-lower alkyl) -amines, such as N, N-dimethyl-N- (2-hydroxyethyl) amine or tris- (2-hydroxyethyl) amine; N-methyl-D-glucamine; ethyl amino acids such as arginine, lysine, and the like.

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

Application method

In one aspect, a method is provided for treating a patient or subject undergoing or having undergone cancer therapy or undergoing or having undergone bone marrow transplantation and/or having been acutely exposed to a myelosuppressive dose of radiation; the method comprises administering to the patient a therapeutically effective amount of a recombinant human GM-CSF protein of the invention or a pharmaceutical composition thereof. In embodiments, the patient is treated by modulating gram Long Kuozeng, survival, differentiation and activation status of hematopoietic progenitor cells. In embodiments, the patient is treated by modulating the bone marrow mononuclear cell lineage, by promoting proliferation of megakaryocytes and erythroid progenitors. In embodiments, the patient is treated by modulating hematopoietic progenitor cells, by stimulating the survival, proliferation and activation of neutrophils, macrophages and/or dendritic cells. In embodiments, the patient is treated after bone marrow transplantation by modulating hematopoietic progenitor cells, by stimulating the survival, proliferation and activation of neutrophils, macrophages and/or dendritic cells.

In one aspect, a method of treatment is provided, comprising administering to a patient a therapeutically effective amount of a recombinant human GM-CSF protein of the invention, or a pharmaceutical composition thereof, or contacting a cell with an effective amount of a pharmaceutical composition described herein and administering a therapeutically effective amount of the cell, wherein the therapy: accelerating neutrophil recovery and/or reducing the incidence of infection following induction chemotherapy; mobilizing hematopoietic progenitor cells into peripheral blood for collection and transplantation by leukopenia; accelerating bone marrow reconstitution after autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation; treatment of delayed neutrophil recovery or transplantation failure following autologous or allogeneic bone marrow transplantation; and/or hematopoietic syndrome (H-ARS) for the treatment of acute radiation syndrome.

In one aspect, there is provided a method for treating a viral infection, the method comprising: administering to a patient in need thereof an effective amount of a composition comprising the recombinant human GM-CSF protein of the invention or a pharmaceutical composition comprising the same.

In embodiments, the viral infection is an influenza infection, optionally selected from influenza a, B, C and D virus infection.

In embodiments, the viral infection is a coronavirus infection. In embodiments, the coronavirus is a type B coronavirus, optionally selected from the group consisting of 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 a type A coronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

Coronaviruses are members of the coronaviridae family, including the recently known respiratory pathogens of human-invading coronaviruses b and a coronaviruses a. The family Coronaviridae includes coronaviruses such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1 and HCoV-OC43. Coronaviruses of type A include, for example, HCoV-NL63 and HCoV-229E.

Coronaviruses invade cells via "spike" surface glycoproteins responsible for virus recognition of angiotensin converting enzyme 2 (ACE 2), a transmembrane receptor on mammalian hosts that promotes viral entry into host cells. Zhou et al A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020. New coronavirus infection (COVID-19) caused by SARS-CoV-2

Is a novel virus believed to originate from bat. Covd-19 causes severe respiratory distress and this RNA virus strain has been the reason for the recent outbreak that has been declared a major threat to public health and global emergency. Phylogenetic analysis of the complete genome of SARS-CoV-2 shows that the virus is most closely related (89.1% nucleotide similarity) to a panel of SARS-like coronaviruses (genus B, subgenera of sand Bei Bingdu). Wu et al Nature,2020, 2/3.

SARS-CoV-2 is an enveloped single-stranded RNA virus that encodes a "spike" protein (also known as S protein), a surface glycoprotein that mediates binding to cell surface receptors; intact membrane proteins; envelope proteins and nucleocapsid proteins. The S protein comprising the S1 and S2 subunits is a trimeric class I fusion protein that is present in a pre-fusion 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 by reference in its entirety. The structure of SARS-CoV-2spike protein in the pre-fusion conformation has been found. See Daniel et al, cryo-EM structure of the SARS-CoV-2spike in the prefusion conformation.Science,2020, month 2, 19, which is incorporated herein by reference in its entirety.

Phylogenetic analysis of the complete genome of SARS-CoV-2 (GenBank accession number: MN 908947) shows that the virus is most closely related (89.1% nucleotide similarity) to a panel of SARS-like coronaviruses (genus B, subgenera of sand Bei Bingdu). Wu et al, 2/3/2020, which is incorporated herein by reference in its entirety.

SARS-CoV-2 has spike surface glycoprotein, membrane glycoprotein M, envelope protein E and nucleocapsid phosphoprotein N. The complete genome of the SARS-CoV-2 coronavirus (29903 nucleotides, single stranded RNA) is described in NCBI database as GenBank reference sequence: MN908947. The coronavirus protein may be selected from the group consisting of: coronavirus spike protein (GenBank reference sequence: QHD 43416), coronavirus membrane glycoprotein M (GenBank reference sequence: QHD 43419), coronavirus envelope protein E (GenBank reference sequence: QHD 43418), coronavirus nucleocapsid phosphoprotein E (GenBank reference sequence: QHD 43423).

In embodiments, the methods prevent or reduce the development of Acute Respiratory Distress Syndrome (ARDS) in a patient.

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

In embodiments, the patient is hypoxic. In embodiments, the patient has respiratory distress. In embodiments, the method improves oxygenation in a patient. In embodiments, the methods prevent or reduce the transition from respiratory distress to cytokine imbalance in a patient. In embodiments, the method reverses or prevents cytokine storm. In embodiments, the method reverses or prevents cytokine storm in the lung or whole body. In embodiments, the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphoproliferative disorder. In embodiments, the methods reverse or prevent the overproduction of one or more inflammatory cytokines. In embodiments, the inflammatory cytokine is one or more of IL-6, IL-1, an IL-1 receptor antagonist (IL-1 ra), IL-2ra, IL-10, IL-18, TNF alpha, interferon gamma, CXCL10, and CCL 7.

In embodiments, the method results in a reduction in viral load in the patient relative to prior to treatment.

In one aspect, there is provided a method for treating or preventing a viral infection in a subject in need thereof, the method comprising providing plasma from a donor subject who has recovered from the viral infection, the plasma comprising IgG, igM and/or IgA antibodies to the virus causing the infection, and the donor subject having been treated with a recombinant human GM-CSF protein as described herein to stimulate production of the antibodies; and administering the plasma to a subject in need thereof. In one aspect, there is provided a method for treating or preventing a viral infection in a subject in need thereof, the method comprising: administering the recombinant human GM-CSF proteins described herein to a donor subject that has recovered from a viral infection; isolating from the donor subject a plasma comprising IgG, igM, and/or IgA antibodies to the virus causing the infection; and administering the plasma to a subject in need thereof.

In embodiments, such methods provide passive immunity against a virus to a subject in need thereof.

In embodiments, igG, igM, and/or IgA antibodies specifically bind to viral antigens. In embodiments, igG, igM, and/or IgA antibodies neutralize viruses. In embodiments, igG, igM, and/or IgA antibodies prevent or reduce infection of cells by a virus.

In embodiments, the viral infection is selected from the group consisting of a coronavirus infection, optionally from the group consisting of 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 a coronavirus infection of type A, optionally from HCoV-NL63 and HCoV-229E infection.

In embodiments, the coronavirus infection is Severe Acute Respiratory Syndrome (SARS).

In embodiments, the coronavirus infection is or is associated with coronavirus disease (covd-19).

In embodiments, the viral infection is an influenza infection, optionally selected from influenza a, B, C and D virus infection. In embodiments, the influenza infection is pandemic 2009 influenza a (H1N 1) or avian influenza a (H5N 1).

In embodiments, the donor subject is positive for the viral infection test prior to recovery. In embodiments, the donor subject has resolved symptoms of the viral infection prior to donation. In embodiments, the donor subject is positive for an antibody test against the virus using a serological test. In embodiments, the donor subject exhibits a measurable neutralizing antibody titer. In embodiments, the neutralizing antibody titer is at least about 1:160. In embodiments, the plasma is isolated from a blood sample of a donor subject. In embodiments, the plasma is separated by plasmapheresis. In embodiments, the plasma comprises a therapeutically effective amount of IgG, igM, and/or IgA antibodies to the virus causing the infection.

Combination therapy and additional therapeutic agents

In various embodiments, the pharmaceutical compositions of the present invention are co-administered in combination with additional agents. The co-administration may be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and GM-CSF of the present invention are administered simultaneously to the subject. As used herein, the term "simultaneously" means that the additional therapeutic agent and GM-CSF are administered at intervals 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. Administration of the additional therapeutic agent and GM-CSF may be performed by simultaneous administration of a single formulation (e.g., a formulation comprising the additional therapeutic agent and GM-CSF composition) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the GM-CSF composition).

Co-administration does not require simultaneous administration of the therapeutic agents if the administration of the additional therapeutic agent and GM-CSF is timed such that the pharmacological activities of the additional therapeutic agent and GM-CSF overlap in time, thereby exerting a combined therapeutic effect. For example, additional therapeutic agents and targeting moiety GM-CSF compositions may be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and GM-CSF are administered at intervals of greater than about 60 minutes. For example, the time interval between sequential administration of additional therapeutic agents and GM-CSF may be greater than about 60 minutes, greater than about 2 hours, greater than about 5 hours, greater than about 10 hours, greater than about 1 day, greater than about 2 days, greater than about 3 days, greater than about 1 week, greater than about 2 weeks, or greater than about one month. The optimal time of administration will depend on the metabolic rate, excretion rate and/or pharmacodynamic activity of the additional therapeutic agent being administered and the GM-CSF. Additional therapeutic agents or GM-CSF compositions may be administered first.

Co-administration also does not require administration of the therapeutic agent to the subject by the same route of administration. Rather, each therapeutic agent may be administered by any suitable route (e.g., parenteral or non-parenteral).

In some embodiments, GM-CSF described herein synergistically acts when co-administered with another therapeutic agent. In such embodiments, the targeting moiety GM-CSF composition and the additional therapeutic agent may be administered at a lower dose than the dose employed when the agent is used in the case of monotherapy.

In some embodiments, the additional therapeutic agent is an antiviral drug.

In some embodiments, the additional therapeutic agent is selected from the group consisting of: antiviral agents, such as adefovir, fampicvir, oseltamivir, balanovir Sha Wei, gan Li Siwei, amprenavir, telanavir, saquinavir, nelfinavir, indinavir, darunavir, atazanavir, ipecine, 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; immunomodulators, such as glucocorticoids, IFN-a 2a, IFN-a 2b, IFN-b, pegylated IFN-g, baroretinib, sirolimus, cara Ji Zhushan anti (clazakizumab), canakinumab, XPro1595, tolizumab, sarilumab, stetuximab, adalimumab, eculizumab, ivermectin, anakinaman, primeverin (prezcobix), xiphoxim, fingolimod, methylprednisolone, leronimab, thalidomide, MK-2206, nicolamide, nitazoxanide, chloroquine or hydroxychloroquine; antibiotics such as colimycin, briolacin, 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, zotagifen, methotrexate, lenalidomide, anti-VEGF-A and intravenous immunoglobulin (IVIG). For example, in embodiments, any of these additional therapeutic agents may be used in the context of SARS-CoV-2 infection.

In some embodiments, the additional therapeutic agent is selected from the group consisting of fampride Weila, peramivir, zanamivir, oseltamivir phosphate, balo Sha Weima boside, wu Minuo, amantadine hydrochloride, adapalamine (adapamine), LASAG/BAY81-87981, celecoxib, etanercept, metformin, gemcitabine, dapivilin, trimetinib, lisinopril, naproxen, nalidixic acid, dorzolamide, lu Suoti, midodrine, diltiazem; statins, including atorvastatin, nitazoxanide; PPAR antagonists, including gemfibrozil. For example, in embodiments, any of these additional therapeutic agents may be used in the context of influenza infection.

Preparation method

In an aspect, there is provided a method of preparing a recombinant producing a composition comprising recombinant human GM-CSF, the method comprising: (a) Obtaining a cell or extract thereof transfected with a nucleic acid encoding a recombinant human GM-CSF comprising an amino acid sequence having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID No. 1 or SEQ ID No. 2 and having a substitution or deletion at positions N37, G38 and/or T39 or corresponding thereto as described herein; (b) Purifying GM-CSF from the transfected yeast cells using one or more HPLC columns, wherein purification is performed in the absence of an organic solvent (such as, but not limited to, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid, and/or n-propanol); and (c) collecting purified GM-CSF that is substantially free of hyperglycosylated, e.g., high mannose, forms of GM-CSF.

In embodiments, the cell is a prokaryotic or eukaryotic host cell, such as a yeast, mammalian, bacterial, insect, algal, or plant cell.

Suitable prokaryotic host cells include bacterial cells (e.g., E.coli, B.subtilis, mycobacterium, M.tuberculosis, or other suitable bacterial cells) and archaebacterial cells (e.g., methanococcus jannaschii and Methanococcus marinus).

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

Suitable fungal host cells include, but are not limited to: ascomycota, basidiomycota, deuteromycota, zygomycota, and incompletely belonging to the class of the fungus.

Suitable yeast host cells include, but are not limited to: candida, hansenula, saccharomyces, schizosaccharomyces, kluyveromyces, and yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, saccharomyces cerevisiae, saccharomyces carlsbergensis, saccharomyces diastaticus, saccharomyces nodosum (Saccharomyces norbensis), kluyveromyces, schizosaccharomyces pombe, kluyveromyces lactis, candida albicans, or yarrowia lipolytica.

Suitable filamentous fungal host cells include, for example, any filamentous form of the phylum Eumycotina and Oomycota. In embodiments, the filamentous fungal host cell may be a cell of the following species: acremonium (Achlya), acremonium (Aspergillus), aureobasidium (Aureobasidium), thiobacillus (Bjerkanadra), ceriporiopsis (Ceriporiopsis), cephalosporium (Cephalosporium), mortierella (Chrysosporium), cavity (Cochliobius), corynamia (Corynascus), convolvulus (Cryponectria), crypocroctis (Cryproctis), coptis (Cryptoccus), coprinus (Coprinus), coriolus (Coriolus), diplodia (Diplodia), endochiosis, fusarium (Fusarium), gibbelopsis (Gibbelopsis), myxomycelia (Gliocladium), hymenomyces (Humicola), hypomycelial (mycelial) (e.g., myceliophthora thermophila Myceliophthora thermophila, mucor (Mucor), neurospora (Neurospora), penicillium (Penicillium), acremonium (Podospora), neurospora (Phlebia), rumex (Piromyces), pyricularia (Pyricularia), rhizomucor (Rhizomucor), rhizopus (Rhizopus), schizophyllum (Schizophyllum), acremonium (Scytalidium), sporotrichum (Sporotrichum), penicillium (Talaromyces), thermomyces (Thermoascus), thielavia, tramata, curvularia (Topocladium), trichoderma, verticillium (Verticillium), volvariella (Volvariella) or either sexual or asexual, and synonyms or taxonomic equivalents thereof. In one embodiment, the filamentous fungus is selected from the group consisting of: aspergillus nidulans, aspergillus oryzae, aspergillus sojae and Aspergillus niger. In one embodiment, the filamentous fungus is Aspergillus niger.

In embodiments, the cell is a yeast cell, such as, but not limited to, saccharomyces cerevisiae.

In embodiments, the cell is a mammalian cell, such as, but not limited to, chinese Hamster Ovary (CHO) cells.

In embodiments, production of the recombinant protein in mammalian cells increases the expression level of the recombinant protein as compared to production using non-mammalian cells.

In embodiments, the method further comprises formulating the purified GM-CSF for injection, e.g., subcutaneous or intravenous injection.

Sequence(s)

SEQ ID NO. 1 is wild type GM-CSF. The site of substitution or deletion of the invention is underlined and bolded:

SEQ ID NO. 2 is a sauce pavilion. The site of substitution or deletion of the invention is underlined and bolded:

definition of the definition

The following definitions are used in connection 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.

An "effective amount" is an amount effective to treat or reduce a coronavirus infection when used in combination with an agent effective to treat a coronavirus infection.

As used herein, "a/an" or "the" may mean one or more than one. In addition, when used in conjunction with a reference numeral designation, the term "about" means that the reference numeral designation adds or subtracts up to 10% of the reference numeral designation. For example, the language "about 50" encompasses a range of 45 to 55.

As mentioned herein, all compositional percentages are by weight of the total composition unless otherwise specified. As used herein, the word "comprise" and variations thereof are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and variations thereof are intended to be non-limiting such that recitation of an embodiment that may or may include certain elements or features does not exclude other embodiments of the inventive technique that do not include those elements or features.

Although the term "comprising" is used herein as a synonym for terms such as including, containing, or having, etc., the description and claims of the present invention, alternative terms such as "consisting of … …" or "consisting essentially of … …" may alternatively be used to describe the present invention or embodiments thereof.

The invention is further illustrated by the following non-limiting examples.

Examples

Example 1: cloning and purification of GM-CSF mutants

Fermentation and processing of the protein GM-CSF product without or without mutations of the invention is performed in the absence of an organic solvent (such as, but not limited to, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid and/or n-propanol) for removing the hyperglycosylated peaks and/or a reverse phase C4 HPLC column for purifying and/or removing the hyperglycosylated peaks.

Importantly, organic solvents and reverse phase C4 HPLC are used to produce GM-CSF lacking the mutations of the invention, e.g., to remove large hyperglycosylated form peaks from the three glycoforms (see FIG. 1). Such organic solvents are not used for the mutants described herein, which avoids the need to use reverse phase C4 HPLC, which aims to remove these organic solvents.

Example 2: biochemical assay of GM-CSF mutants

The biochemical identity of rhu GM-CSF was confirmed by Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis (FIG. 2). Purified rhu GM-CSF protein samples were diluted in Tris-glycine SDS sample buffer. Diluted rhu GM-CSF samples and reference standards were denatured by heating at 100 ℃ for 5 minutes in a heated block. The reference standard and rhu GM-CSF samples were loaded onto a preformed 20% SDS-PAGE gel along with the molecular weight markers. The loaded gel was electrophoresed at a constant voltage to isolate the protein and then incubated in coomassie G-250 staining to visualize the protein. rhu GM-CSF migrates into 3 bands on SDS-PAGE. Supernatants were run on SDS-PAGE (4 ug/lane) and compared to molecular weight ladders and reference standards.

Western blot analysis was performed to confirm protein identity (fig. 2). Purified rhu GM-CSF protein samples and reference standards were prepared for SDS-PAGE as previously described. The reference standard and rhu GM-CSF samples were loaded onto a pre-fabricated 20% SDS-PAGE along with pre-stained molecular weight markers. The loaded gel was subjected to electrophoresis at a constant voltage to separate proteins, which were then transferred from the gel onto a nitrocellulose membrane. Nitrocellulose membranes were then blotted with mouse anti-human GM-CSF monoclonal antibodies, and goat anti-mouse IgG conjugated with alkaline phosphatase was then used for immunodetection. The bands were visualized using alkaline phosphatase developer. All supernatants were blotted positive for GM-CSF (positive bands in the GM-CSF region compared to the reference standard) (FIG. 2).

Reversed phase HPLC was used to determine the percentage distribution of rhu GM-CSF glycoform (FIG. 3). Neither organic solvents nor reverse phase C4 HPLC columns were used. Single point mutant T39A (alanine for threonine at position 39) and N37Q (glutamine for asparagine at position 37) were tested in a reverse phase HPLC assay to determine potency and glycoform. The assay uses a calibration curve made from a reference standard to generate the effective value (mg/ml). This procedure resolved rhu GM-CSF glycosylation variants into three major glycoform groups. The test sample rhu GM-CSF concentration results were interpolated from six external standard calibration curves prepared from GM-CSF reference standards. Integrating and quantifying four target peaks; the composition of each is described below:

Peak 1 = GM-CSF related impurity (oxidation).

Peak 2 = N-and (n+o) linked sugar

Peak 3 = O-linked saccharide

Peak 4 = non-glycosylated GM-CSF

The assay separates the residual process components and hyperglycosylated material from the product peaks to obtain a glycoform profile (peaks 2-4). The product peaks are reported as normalized so as not to take into account hyperglycosylated species. T39A has about 6% hyperglycosylation and N37Q has about 6.6% hyperglycosylation. Both samples had a lower percentage of high glycosylation of GM-CSF without mutation (about 40%, see figure 1). The hyperglycosylated species are typically removed by chromatography using organic solvents such as, but not limited to, acetonitrile, trifluoroacetic acid (TFA), pyridine, acetic acid and/or n-propanol, and reverse phase C4 HPLC columns for purification purposes, and these mutants are interesting because, among other things, they may be able to eliminate process steps, which would save time, resources and use less hazardous solvents in the purification process.

Reverse phase HPLC was also used to determine the GM-CSF glycoform of BDS LEUKINE compared to CHO-expressed mutant GM-CSF (FIG. 4). Size exclusion chromatography was also used to determine the molar mass distribution (fig. 5). Single point mutant N37Q (asparagine at glutamine substitution position 37) and BDS LEUKINE were tested in reverse phase HPLC assay and SEC analysis to determine titers (fig. 6) and glycoforms (fig. 4 and 5). BDS LEUKINE is produced using Saccharomyces yeast cells, and mutant GM-CSF is produced using mammalian CHO cells. The assay uses a calibration curve made from a reference standard to generate the effective value (mg/ml). This procedure resolved rhu GM-CSF glycosylation variants into three major glycoform groups. The test sample rhu GM-CSF concentration results were interpolated from six external standard calibration curves prepared from GM-CSF reference standards. Similar to BDS LEUKINE, CHO-expressed mutant GM-CSF was monomeric (fig. 5), and both samples showed multiple glycoforms (fig. 4). Production in CHO cells increased the potency of the recombinant protein compared to production in yeast (fig. 6). BDS LEUKINE refers to wild type GM-CSF, SEQ ID NO:1, without T39A or N37Q amino acid substitutions within the sequence.

Example 3: functional assays for GM-CSF mutants

The efficacy of rhu GM-CSF was achieved using the following protocolTF-1 cells were human erythroleukemia cell lines that proliferated in response to GM-CSF as measured by bioassay of TF-1 cells (FIG. 7). Reference standard (rhu GM-CSF), control and test samples were serially diluted in triplicate in assay medium and added to three separate 96-well plates. TF-1 cells in suspension were then added and the mixture incubated at 37℃for 69.5-72 hours. After addition of fluorescent dye (ALAMARBLUE, THERMO), the plates were incubated at 37℃for 6.6-8 hours. TF-1 cell proliferation was then measured in a fluorescent plate reader. Single point T39A and N37Q, as well as GM-CSF without T39A or N37Q mutation (LEUKINE) were tested in the TF-1 bioassay. Specific Activity (SA) in International units per milligram (IU/mg) was determined from the standard curve using a reference standard (Shagrastim-rhuGM-CSF). Both mutants showed similar activity to LEUKINE and SA was determined to be 9.13x10 ⁶ (T39A) and 7.48x 10 ⁶ (N37Q) in contrast to 8.14x10 for LEUKINE ⁶ IU/mg：

/>

Surprisingly, these amino acid substitutions, while being drastic (e.g., without wishing to be bound by theory, significantly reduce the side chain length for T39A-and increase the side chain length for N37Q), do not affect biological activity while allowing for easier manufacturability.

In addition, the efficacy of mutant rhu GM-CSF was measured using a bioassay with TF-1 cells, a human erythroleukemia cell line that proliferated in response to GM-CSF. Mutant rhu GM-CSF with a single amino acid at position N37 grown in CHO cells significantly enhanced the activity of the recombinant protein compared to 3 different batches of BDS LEUKINE (novel material of LEUKINE BDS, LEUKINE BDS stored for 12 months at 2-8 ℃ and LEUKINE stored for 48 months at-70 ℃) grown in yeast cells, saccharomyces stored using different conditions (fig. 8). BDS LEUKINE refers to wild type GM-CSF, SEQ ID NO:1, having NO T39A or N37Q amino acid substitution within its sequence.

Equivalent scheme

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

Incorporated by reference

All patents and publications cited herein are incorporated by reference in their entirety.

As used herein, all headings are for organization only and are not intended to limit the disclosure in any way. The contents of any single portion may apply equally to all portions.

Sequence listing

<110> partner treatment Co., ltd

M.Fisdeluxe (FELDHAUS, michael)

J.Joster (YOST, jeffrey)

<120> granulocyte macrophage colony stimulating factor mutant

<130> PNR-003PC/127114-5003

<150> US 63/105,425

<151> 2020-10-26

<150> US 63/177,481

<151> 2021-04-21

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> synthetic sequence

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Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val

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Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr

20 25 30

Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp

35 40 45

Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln

50 55 60

Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met

65 70 75 80

Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys

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Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp

100 105 110

Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

<210> 2

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> synthetic sequence

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Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val

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Asn Ala Ile Gln Glu Ala Leu Arg Leu Leu Asn Leu Ser Arg Asp Thr

20 25 30

Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp

35 40 45

Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln

50 55 60

Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met

65 70 75 80

Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys

85 90 95

Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp

100 105 110

Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

Claims

1. A composition comprising a recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) protein comprising a nucleotide sequence having at least about 97% identity to SEQ ID No. 1 or SEQ ID No. 2 and a substitution or deletion at or corresponding to position N37, E38 and/or T39.

2. The recombinant protein according to claim 1, wherein an amino acid at position N37 or a position corresponding thereto is a polar and charge neutral hydrophilic amino acid.

3. The recombinant protein of claim 2, wherein the polar and charge neutral hydrophilic amino acid is selected from the group consisting of glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

4. The recombinant protein of claim 3, wherein the polar and charge neutral hydrophilic amino acid is glutamine (Q).

5. The recombinant protein according to claim 1, wherein an amino acid at position E38 or a position corresponding thereto is a hydrophobic aliphatic amino acid and/or is not proline.

6. The recombinant protein according to claim 5, wherein said hydrophobic aliphatic amino acid is selected from the group consisting of alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

7. The recombinant protein according to claim 1, wherein an amino acid at position T39 or a position corresponding thereto is a hydrophobic aliphatic amino acid.

8. The recombinant protein according to claim 7, wherein said hydrophobic aliphatic amino acid is selected from the group consisting of alanine (a), glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V).

9. The recombinant protein according to claim 8, wherein said hydrophobic aliphatic amino acid is alanine (a).

10. The recombinant protein of any one of claims 1-9, wherein the composition binds to and/or activates the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R- α or CSF 2R).

11. The recombinant protein of claim 10, wherein the GM-CSF-R- α is expressed on the surface of a cell.

12. The recombinant protein according to claim 11, wherein said cell is a hematopoietic progenitor cell.

13. The recombinant protein according to claim 12, wherein said hematopoietic progenitor cells are immune cells.

14. The recombinant protein according to claim 12, wherein said hematopoietic progenitor cells are irradiated.

15. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is soluble.

16. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is functionally similar to a saxagliptin.

17. The recombinant protein according to any one of the preceding claims, wherein said recombinant human GM-CSF comprises a plurality of molecular forms.

18. The recombinant protein according to claim 17, wherein said molecular form is selected from the group consisting of a non-glycosylated form, an O-glycosylated form, an N-glycosylated form, and an n+o glycosylated form.

19. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of high mannose glycosylated forms.

20. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is resolved into three peaks when quantified by reverse phase high performance liquid chromatography (RP-HPLC).

21. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF does not provide a significant peak at a retention time of less than about 20 minutes when quantified by reverse phase high performance liquid chromatography (RP-HPLC).

22. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of high mannose glycosylated forms when purified without the use of an organic solvent.

23. The recombinant protein of any one of the above claims, wherein the recombinant human GM-CSF is substantially free of high mannose glycosylated forms, optionally when purified without use of a reverse phase C4 HPLC column.

24. A nucleic acid molecule encoding the recombinant human GM-CSF of any one of the preceding claims.

25. The nucleic acid of claim 24, wherein the nucleic acid molecule has a codon optimized sequence.

26. A non-human host cell expressing the nucleic acid molecule of claim 23 or 24.

27. The host cell of claim 26, wherein the host cell is a yeast cell.

28. The host cell of claim 27, wherein the yeast cell is a non-methylotrophic yeast cell.

29. The host cell of claim 28, wherein the host cell is a saccharomyces cerevisiae cell.

30. A pharmaceutical composition comprising the recombinant human GM-CSF of any one of the preceding claims and a pharmaceutically acceptable excipient or carrier.

31. A method of treating a patient or subject undergoing or having undergone cancer therapy or undergoing or having undergone bone marrow transplantation and/or having been acutely exposed to a myelosuppressive dose of radiation; the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 30.

32. The method of claim 31, wherein the patient is treated by modulating gram Long Kuozeng, survival, differentiation and activation status of hematopoietic progenitor cells.

33. The method of claim 31, wherein the patient is treated by modulating a bone marrow monocyte lineage, by promoting proliferation of megakaryocytes and erythroid progenitors.

34. The method of claim 31, wherein the patient is treated by modulating hematopoietic progenitor cells, by stimulating the survival, proliferation and activation of neutrophils, macrophages and/or dendritic cells.

35. The method of claim 31, wherein the patient is treated after bone marrow transplantation by modulating hematopoietic progenitor cells, by stimulating the survival, proliferation and activation of neutrophils, macrophages and/or dendritic cells.

36. A method of treatment, the method of treatment comprising

Administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 30, or

Contacting a cell with an effective amount of the pharmaceutical composition of claim 30 and administering a therapeutically effective amount of the cell,

wherein the therapy is:

accelerating neutrophil recovery and/or reducing the incidence of infection following induction chemotherapy;

mobilizing hematopoietic progenitor cells into peripheral blood for collection and transplantation by leukopenia;

accelerating bone marrow reconstitution after autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation;

treatment of delayed neutrophil recovery or transplantation failure following autologous or allogeneic bone marrow transplantation; and/or

Hematopoietic syndrome (H-ARS) for the treatment of acute radiation syndrome.

37. A method for treating a coronavirus infection, the method comprising: administering to a patient in need thereof an effective amount of a composition comprising the recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) protein of any one of claims 1-22.

38. The method of claim 36, wherein the coronavirus is a type b coronavirus, optionally selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, middle east respiratory syndrome-coronavirus (MERS-CoV), HCoV-HKU1, and HCo V-OC43.

39. The method of claim 37, wherein the coronavirus is a coronavirus type a, optionally selected from HCoV-NL63 and HCoV-229E.

40. The method of claim 39, wherein the coronavirus is SARS-CoV-2.

41. The method of claim 40, wherein the patient has a COVID-19.

42. The method of any one of claims 37-41, wherein the patient has one or more of fever, cough, shortness of breath, diarrhea, upper respiratory symptoms, lower respiratory symptoms, pneumonia, and acute respiratory syndrome.

43. The method of any one of claims 37-42, wherein the patient is hypoxic.

44. The method of any one of claims 37-43, wherein said patient has respiratory distress.

45. The method of any one of claims 37-44, wherein said method prevents or reduces the development of Acute Respiratory Distress Syndrome (ARDS) in said patient.

46. The method of any one of claims 37-45, wherein the method improves oxygenation in the patient.

47. The method of any one of claims 37-46, wherein said method prevents or reduces the transition of said patient from respiratory distress to cytokine imbalance.

48. The method of any one of claims 37-47, wherein the method reverses or prevents cytokine storm.

49. The method of claim 48, wherein the method reverses or prevents cytokine storm in the lung or whole body.

50. The method of claim 48 or 49, wherein the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome and hemophagocytic lymphoproliferative disorder.

51. The method of claim 48 or 49, wherein said method reverses or prevents overproduction of one or more inflammatory cytokines.

52. The method of claim 51, wherein the inflammatory cytokine is one or more of IL-6, IL-1 receptor antagonist (IL-1 ra), IL-2ra, IL-10, IL-18, TNF alpha, interferon gamma, CXCL10, and CCL 7.

53. The method of any one of claims 37-52, wherein the method results in a reduction in viral load in the patient relative to prior to treatment.

54. A method for treating or preventing a viral infection in a subject in need thereof, the method comprising:

Providing plasma from a donor subject who has recovered from the viral infection,

the plasma comprises IgG, igM and/or IgA antibodies against the virus causing the infection, and

the donor subject has been treated with the recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) protein of any one of claims 1-22 to stimulate the production of the antibodies; and

administering the plasma to the subject in need thereof.

55. A method for treating or preventing a viral infection in a subject in need thereof, the method comprising:

administering the recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) protein of any one of claims 1-22 to a donor subject that has recovered from the viral infection;

classifying plasma from the donor subject, the plasma comprising IgG, igM, and/or IgA antibodies to the virus causing the infection; and

administering the plasma to the subject in need thereof.

56. The method of claim 54 or 55, wherein the method provides passive immunization against the virus to the subject in need thereof.

57. The method of any one of claims 54-56, wherein the IgG, igM, and/or IgA antibodies specifically bind to a viral antigen.

58. The method of claim 57, wherein the IgG, igM, and/or IgA antibodies neutralize the virus.

59. The method of claim 57 or 58, wherein the IgG, igM and/or IgA antibodies prevent or reduce infection of cells by the virus.

60. The method of any of claims 54-59, wherein the viral infection is selected from a group consisting of a coronavirus infection, optionally from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a severe acute respiratory syndrome coronavirus (SARS-CoV-1), a middle east respiratory syndrome-coronavirus (MERS-CoV), an HCoV-HKU1, and an HCoV-OC43 infection.

61. The method of any one of claims 54-60, wherein the viral infection is selected from a coronavirus infection of type a, optionally from an HCoV-NL63 and an HCoV-229E infection.

62. The method of claim 61, wherein the coronavirus infection is Severe Acute Respiratory Syndrome (SARS).

63. The method of claim 61, wherein the coronavirus infection is or is associated with coronavirus disease (COVID-19).

64. The method of any one of claims 54-63, wherein the viral infection is an influenza infection, optionally selected from the group consisting of an influenza a, B, C, and D virus infection.

65. The method of claim 64, wherein the influenza infection is pandemic 2009 influenza a (H1N 1) or avian influenza a (H5N 1).

66. The method of any one of claims 54-65, wherein the donor subject is positive for a viral infection test prior to recovery.

67. The method of any one of claims 54-66, wherein the donor subject has resolved symptoms of a viral infection prior to donation.

68. The method of any one of claims 54-67, wherein the donor subject is positive for an antibody test against the virus using a serological test.

69. The method of any one of claims 54-68, wherein said donor subject exhibits a measurable neutralizing antibody titer.

70. The method of claim 69, wherein the neutralizing antibody titer is at least about 1:160.

71. The method of any one of claims 54-70, wherein the plasma is isolated from a blood sample from the donor subject.

72. The method of claim 71, wherein the plasma is isolated via plasmapheresis.

73. The method of any one of claims 54-72, wherein said plasma comprises a therapeutically effective amount of said IgG, igM, and/or IgA antibodies to a virus that causes said infection.

74. A method of preparing a recombinant that produces a composition comprising recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF), the method comprising:

(a) Obtaining a cell transfected with a nucleic acid encoding a recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) comprising an amino acid sequence having at least about 97% identity to SEQ ID No. 2 and having a substitution or deletion at positions N37, G38 and/or T39 or positions corresponding thereto;

(b) Purifying the GM-CSF from the transfected cells using one or more HPLC columns, wherein the purifying is performed in the absence of an organic solvent; and

(c) Collecting said purified GM-CSF, said purified GM-CSF being substantially free of high mannose glycosylated forms of GM-CSF.

75. The method of claim 74, wherein the cell is a yeast cell.

76. The method of claim 75, wherein the yeast cell is Saccharomyces cerevisiae.

77. The method of claim 74, wherein the cell is a mammalian cell.

78. The method of claim 77, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

79. The method of claim 78, wherein transfection of mammalian cells, such as CHO cells, increases the expression level of the recombinant protein during production of the recombinant protein as compared to production methods using non-mammalian cells.

80. The recombinant protein of claim 74, wherein the recombinant human GM-CSF exhibits enhanced function as compared to a sauce pavilion.

81. The recombinant protein of claim 1, produced by transfection of mammalian cells such as CHO cells, wherein the recombinant human GM-CSF exhibits enhanced function as compared to that of sagrastim.

82. A composition comprising a recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) protein comprising an amino acid sequence having at least about 97% identity to SEQ ID NO. 1 or SEQ ID NO. 2 and having an N37Q substitution or position corresponding thereto,

wherein the GM-CSF is capable of isolation without significant hyperglycosylated species and without the need for organic solvent purification.