WO2007009208A1 - Gm-csf humain modifie au poly(ethylene glycol) qui presente une activite biologique accrue - Google Patents

Gm-csf humain modifie au poly(ethylene glycol) qui presente une activite biologique accrue Download PDF

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WO2007009208A1
WO2007009208A1 PCT/CA2006/000885 CA2006000885W WO2007009208A1 WO 2007009208 A1 WO2007009208 A1 WO 2007009208A1 CA 2006000885 W CA2006000885 W CA 2006000885W WO 2007009208 A1 WO2007009208 A1 WO 2007009208A1
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csf
peg
human
molecule
kda
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PCT/CA2006/000885
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Darin Lee
Don Stewart
Vadim Tsvetnitsky
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Cangene Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the instant invention relates to a chemical modification of granulocyte- macrophage colony-stimulating factor (GM-CSF), by which the chemical and/or physiological properties of GM-CSF can be changed.
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • Recombinant human GM-CSF is known in the art and used to treat several disorders including neutropenia following radiotherapy or chemotherapy. As with most cytokines, human GM-CSF has a short half-life in circulation, requiring repeated injections.. Additionally, recombinant human GM-CSF has been shown to be immunogenic and antigenic, leading to the generation of inhibitory antibodies.
  • Regramostim produced in CHO cells, has the full-length native human GM-CSF peptide sequence, and has the most complete (closest to human) glycosylation profile. As a consequence of its full glycosylation, Regramostim has increased half-life but a significantly reduced bioactivity compared to other GM-CSF products (Hussein et al, 1995).
  • Sargramostim (LeukineTM), produced in the yeast Saccharomyces cereviseae, differs from the native human GM-CSF amino acid sequence by virtue of a substitution of Leucine at position 23. This change ameliorates a protease degradation problem in the yeast production system (US 5,393,870). Even a single amino acid change, however, can be implicated in increased immunogenicity of proteins in general and is proposed as a causative factor in yeast GM-CSF immunogenicity in particular (Rini et al, 2005). Sargramostim, by virtue of its production in yeast, is somewhat glycosylated. As a consequence of its glycosylation, Sargramostim has significantly reduced bioactivity in vivo compared to non-glycosylated GM-CSF (Hussein et al, 1995).
  • Molgramostim (LeucomaxTM), produced in E.coli, differs from the native human GM-CSF amino acid sequence by virtue of deletion of the six N-terminal amino acids, as well as the addition of an N-terminal methionine residue.
  • Molgramostim As a bacterial protein product, Molgramostim is unglycosylated, contributing to its superior bioactivity compared to Regramostim ⁇ and Sargramostim (Hussein et al, 1995). Molgramostim's side-effect profile is claimed to be worse than that of Sargramostim (Dorr, 1993), but it is suggested that this is due to unnecessarily high doses of the more active non-glycosylated form.
  • the observed activity may be the result of the cells reacting to exposure to radiation, lodination is a harsh treatment that often results in modification of a protein's secondary structure.
  • lodination is a harsh treatment that often results in modification of a protein's secondary structure.
  • Sherman et al teach the conjugation of 19 kDa PEG to human GM-CSF by p-Nitrophenyl Carbonate to produce mono-PEGylated and di-PEGylated human GM-CSF. Additionally, Sherman et al conjugated either a 5 kDa or 42 kDa PEG to human GM-CSF by a PEG aldehyde to produce a mono-PEGylated human GM-CSF. Sherman states that human GM-CSF is from Immunex, which produces Sargramostim (LeukineTM) in a yeast expression system. As described above, glycosylated GM-CSF behaves differently than non-glycosylated GM-CSF.
  • DeFrees et al describe a site-directed PEGylated human GM-CSF prepared by expressing non-glycosylated GM-CSF in E. coli, followed by enzymatic GalNAc-glycosylation at specific serine and threonine residues, followed by enzymatic transfer of sialic acid conjugated with linear 2OkDa PEG to the introduced GaINAc residues (primarily at Ser 7 and Ser 9 ).
  • a non-homogenous mixture of singly, doubly and triply-PEGylated GM-CSF was produced by this method, and neither in vitro nor in vivo experiments to demonstrate bioactivity or pharmacokinetic parameters were performed (DeFrees et al, 2006).
  • Doherty et al created analogues of human GM-CSF engineered to contain additional cysteine residues, which were then PEGylated with cysteine-reactive 5kDa, 1OkDa, or 2OkDa linear PEG or 4OkDa branched PEG.
  • the 5, 10 and 2OkDa PEG modifications did not substantially change the in vitro bioactivity of human GM-CSF, while the 4OkDa PEGylation decreased bioactivity.
  • the PEGylated cysteine-bearing GM-CSFs demonstrated increased halflives in rats correlating with the size of the PEG molecules; since human GM-CSF does not function in rodents, no in vivo efficacy data was presented (Doherty et al, 2005).
  • Bossard et al demonstrate the synthesis of human GM-CSF conjugated to a variety of activated PEG molecules, including 4OkDa branched mPEG, 3OkDa linear mPEG, and 2OkDa linear mPEG.
  • the authors show how to prepare and purify PEG-GM-CSF conjugates, but simply claim that in vitro bioactivity of the conjugates has been demonstrated, without quantifying the bioactivity or even describing the assays used.
  • no animal data are presented whatsoever. As such, there is no indication if PEGylated human GM-CSG has any activity in an animal.
  • the present invention is a full length, non-glycosylated human GM-CSF having a single polyethylene glycol molecule covalently attached through an N-terminal amino acid.
  • the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising human GM-CSF having a single polyethylene glycol molecule covalently attached through ah N-terminal amino acid.
  • the present invention is the use of human GM-CSF having a single polyethylene glycol molecule covalently attached through an N-terminal amino acid to increase circulating neutrophils in a primate.
  • FIG.1 shows capillary electrophoretic separation of human GM-CSF PEGylation reaction coupled to zero, one, two or three PEG molecules per protein.
  • FIG. 2 shows a RP-HPLC tracing of purified monoPEGylated human GM- CSF.
  • FIG. 3 shows a RF- HPLC analyses of tryptic peptide digests of A. GM-CSF and B. 20 kDa PEG-GM-CSF.
  • FIG. 4 shows a GM-CSF dependent TF-1 cell proliferation
  • Triangles 20 kDa PEG-GM-CSF; diamonds: NIBSC human GM-CSF standard; squares non-PEGylated GM-CSF.
  • FIG. 5 shows rat serum concentrations of 20 kDa PEGylated GM-CSF (triangles) and nonPEGylated GM-CSF (squares).
  • FIG. 6 shows absolute neutrophil count of monkeys administered 20 kD, 30 kD and 40 kD PEG-GM-CSF on days 1 and 7 post 6.00 cGy x-irradiation.
  • FIG. 7 shows the serum concentration of 20 kD, 30 kD and 40 kD PEG- GM-CSF in the monkeys described in Fig 6.
  • FIG. 8 shows DNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of GM-CSF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • human GM-CSF shall mean an isolated human granulocyte- macrophage colony stimulating factor polypeptide that does not have glycosylation groups attached. Specifically, a human GM-CSF polypeptide substantially having 127 amino acids and the amino acid sequence of mature human granulocyte-macrophage colony stimulating factor as described in US Patent No. 5,891,429 for example as shown in Figure 7.
  • GM-CSF GM-CSF protein that is non-glycosylated and has a single PEG molecule on the N-terminal amino acid.
  • N-terminal pegylated GM-CSF has significantly enhanced potency.
  • human GM-CSF polypeptide examples include expression systems utilizing E. coli, assuming the human GM-CSF polypeptide is expressed as a mature GM- CSF polypeptide having substantially 127 amino acids.
  • Alternate sources for human GM- CSF that may be used in the instant invention include human GM-CSF polypeptides isolated from mammalian or yeast expression system wherein the glycosylation groups are removed from the human GM-CSF polypeptide. Plant and insect cell expression systems may also be used to express human GM-CSF.
  • PEG refers to poly(ethylene glycol) having a chemical formula HO(CH 2 )nCH 2 OH.
  • blocking group as used herein is intended to imply a moiety which when covalently bound to a PEG terminus, is capable of preventing the attachment of an activating group to that terminus during the activation process.
  • blocking groups include monomethoxypoly(ethylene glycol), usually abbreviated as mPEG, which contains an additional monomethoxy group at one end of the polymer (chemical formula CH 3 O - (CH 2 CH 2 O) n - CH 2 CH 2 OH).
  • PEG-GM-CSF refers to granulocyte- macrophage colony-stimulating factor conjugated to poly(ethylene glycol) with a blocking group.
  • the blocking group is part of the PEG molecule and ensures one PEG reacts with one GM-CSF molecule. Without the blocking group, PEG could react at both ends resulting in two GM-CSF molecules being cross-linked by one PEG molecule.
  • the preferred molecular weight of the PEG is between about 20 kDa and about 50 kDa.
  • the term "about” indicates that in. preparations of polyethylene glycol, some molecules will weigh more or less than the stated molecular weight.
  • the mass mentioned before PEG-GM-CSF refers only to the mass of the polymer itself.
  • 20 kDa PEG-GM-CSF refers to an average PEG molecular mass of 20 kiloDaltons.
  • the total molecular mass of the PEG-GM-CSF would be the molecular mass of the PEG (20 kDa), plus the molecular mass of the human GM-CSF (14.5 kDa), for a total of 34.5 kDa.
  • the PEG molecule is less than 20 kDa, then the PEG-GM-CSF is not likely to have a significant increase in half-life in circulation. If the PEG molecule is greater than 50 kDa, then PEG-GM-CSF has decreased biological activity, likely due to steric hindrance of the PEG molecule interfering with GM-CSF binding to its receptor.
  • PEG PEG
  • Nektar Therapeutics San Carlos, CA, US
  • Enzon Pharmaceuticals Bridgewater, NJ, US
  • Sigma-Aldrich Sunbio USA
  • Sunbio USA Orinda, CA, US
  • NOF Corporation NOF Corporation (Tokyo, Japan).
  • PEG may be prepared, for example, as described in U.S. Pat. No. 5,428,128; U.S. Pat. No. 6,127,355; and U.S. Pat. No. 5,880,131. Description of PEGylation reaction
  • the instant application contemplates a PEG molecule bound onto human GM-CSF via a N-terminal reactive group, specifically the alpha amine group at the N- terminus.
  • PEG aldehyde is reacted with human GM-CSF under such conditions that a ' single molecule of PEG is attached preferentially to the' N- terminal amino acid of the human GM-CSF.
  • PEG aldehyde is reacted with human GM-CSF via a reductive amination reaction in the presence of a reducing agent.
  • the resulting secondary amine bond between the mPEG and human GM-CSF that is formed upon reduction is stable under physiological conditions.
  • the site of attachment is at the alpha-terminal amine at the N-terminus of the human GM-CSF.
  • the mPEG aldehyde is mPEG butyraldehyde reacted at pH 5 to 7.5 in the presence of sodium cyanobromohydride.
  • chemistries may be used to chemically attach PEG molecules to the N-terminal amino acid.
  • One alternative that may be used to form a terminal reactive group on monomethoxyPEG (MPEG) is tresyl chloride (2,2,2,-trifluoroethane-sulphonyl chloride), which creates a single free derivatisable OH group.
  • tresyl chloride (2,2,2,-trifluoroethane-sulphonyl chloride)
  • Activation of the hydroxyl group at the end of the polymer opposite to the terminal methoxy group is generally necessary to accomplish efficient protein PEGylation, with the aim being to make the derivatised PEG more susceptible to nucleophilic attack.
  • the attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines can also react (e.g. the N-terminal alpha-amine or the ring amines of histidine) if conditions are favourable.
  • site-specific PEGylation is achieved by introducing a "free" cysteine residue, i.e., a cysteine residue not involved in a disulfide bond, into a target protein using site-directed mutagenesis.
  • the free cysteine residue serves as the attachment point for covalent modification of the protein with a cysteine- reactive PEG molecule.
  • Attachment of the PEG molecule to the free cysteine residue is highly specific because most native cysteine residues in proteins participate in disulfide bonds and are not available for PEGylation using cysteine-reactive PEGs. PEGylation of the cysteine muteins yield a single monoPEGylated species modified at the free cysteine residue.
  • PEG is attached to the GM-CSF polypeptide directly or without an intervening linker.
  • PEG can also be attached to the N-terminal amino acid of GM-CSF polypeptides using a number of different intervening linkers that serve as a terminal reactive group.
  • intervening linkers that serve as a terminal reactive group.
  • U.S. Pat. No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein or polypeptide by a linker can also be produced by reaction of proteins or polypeptides with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1 ,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-.rho.- nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • MPEG-succinimidylsuccinate MPEG activated with 1 ,1'-carbonyldiimidazole
  • MPEG-2,4,5-trichloropenylcarbonate MPEG-.rho.- nitrophenolcarbonate
  • MPEG-succinate derivatives A number of additional polyethylene glycol derivatives and reaction chemistries for attaching PEG to proteins and polypeptides are described in EP 0 714 402.
  • the choice of chemical reaction used to attach the PEG to the N-terminal amino acid of human GM-CSF may affect the choice of the proportion of PEG molecules to GM-CSF molecules used in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally monoPEGylated GM- CSF polypeptides from the reaction mix.
  • the method of obtaining the N-terminally monoPEGylated preparation i.e., separating this moiety from other PEGylated moieties if necessary
  • Selective chemical modification at the N- terminus of the protein may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N- terminus) available for derivatization in a particular protein. Under the appropriate range of reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. Purification and Confirmation of Identity of PEG-GM-CEF
  • a polyethylene glycol-modified human GM-CSF may be purified from a reaction mixture by conventional methods which are used for purification of proteins, such as dialysis, salting-out, ultrafiltration, FPLC (fast protein liquid chromatography protocol), ion- exchange chromatography, gel chromatography and electrophoresis. Ion-exchange chromatography and FLPC are particularly effective in removing unreacted polyethylene glycol and human GM-CSF.
  • a polyethylene glycol-modified human GM-CSF may also be purified from a reaction mixture by partitioning the PEG-GM-CSF in a PEG-containing aqueous biphasic system as described in US Patent No. 6,384,195. Testing in vitro
  • the major forms of in vitro assay used to assess the direct effects of human GM- CSF include cell proliferation assays and assays that assess the ability of the molecule to induce colony formation in progenitor cells.
  • Proliferation In proliferation assays, cells grow and divide in response to human GM- CSF. Proliferation may be measured directly by counting the cells (microscope or automated methods), or indirectly by measuring the uptake of radioactive compounds "such as tritiated thymidine, by the use of chromogenic dyes to quantitate total protein, or by measuring metabolic activity through the use of substrates that form coloured products upon reaction with cellular enzymes (including, but not limited to, the MTT assay and refinements thereof such as the XTT and WST-1 assays, and the Alamar Blue assay).
  • radioactive compounds such as tritiated thymidine
  • chromogenic dyes to quantitate total protein
  • metabolic activity through the use of substrates that form coloured products upon reaction with cellular enzymes (including, but not limited to, the MTT assay and refinements thereof such as the XTT and WST-1 assays, and the Alamar Blue assay).
  • GM-CSF The most common in vitro bioassay of GM-CSF, used to establish activity comparisons to WHO standard GM-CSF, is a standard cell proliferation assay (coluorimetric or tritiated thymidine incorporation) using the human growth factor-dependent cell line TF-1.
  • Other cell lines that may be used to assay GM-CSF mediated proliferation include (bur are not limited to) AML-193 ; B6SUt-A ; BAC1.2F5 ; BCL1 ; Da ; FDCP 1 ; GF-D8 ; GM/SO ; IC-2 ; KG-1 ; MO7E ; NFS-60 ; PT-18 ; TALL-103 ; UT-7.
  • GM-CSF Another test of human GM-CSF biological activity is the colony formation or clonogenic assay.
  • GM-CSF dependent cells are grown in a viscous medium such as soft agar, methylcellulose, plasma gel or fibrin clots, which retards cell movement, allowing the formation of localized cell colonies:
  • the activity of human GM- CSF is assayed by its ability to induce development of differentiated colonies of hematopoietic progenitor cells from bone marrow over period of 7 -14 days.
  • This type of assay demonstrates the ability of human GM-CSF to appropriately determine the lineage along which colony-forming cells differentiate. By performing this assay in a dose- response format, specific activity of human GM-CSF can also be measured.
  • Vivo Testing of PEG-GM-CSF In Vivo Testing of PEG-GM-CSF
  • Animal models appropriate for such testing include, but are not limited to, humans, non-human primates, canines, and rodents (such as rabbits, " rats and mice). Furthermore, the animal models may be healthy, or reflect various disease states, such as diabetes or radiation sickness.
  • GM-CSF In vivo testing of GM-CSF is also helpful in determining its effect on hematopoietic responses, dose response relationships and toxicities. Due to the species specificity of GM-CSF's effects, primate models are necessary for this type of in vivo
  • healthy or irradiated monkeys are injected with GM-CSF and blood cell counts are monitored, including but not limited to circulating leukocytes, granulocytes, monocytes, neutrophils, platelets and erythrocytes.
  • compositions of PEG-GM-CSF may consist of, but are not limited to, liquid or lyophilized preparations preferably suitable for subcutaneous or intravenous administration, and may also include alternate routes of administration including intramuscular, oral or inhalational.
  • a lyophilized formulation for PEG-GM-CSF may include a stabilizer to inhibit denaturation of the PEG-GM-CSF during lyophilization and rehydration, and optionally a buffer, a tonicifying agent and a preservative. However, a buffer and tonicifying agent may be included in the solution utilized to rehydrate the lyophilized PEG-GM-CSF.
  • a liquid, ready for use, PEG-GM-CSF formulation may include a stabilizer to inhibit denaturation of the PEG-GM-CSF during storage or when exposed to shear forces, a tonicifying agent, and optionally a buffer and optionally a preservative if the formulation is for multiple uses.
  • stabilizers include, sugars, amino acids, organic salts, inorganic salts, diluents, buffering agents, surfactants and preservatives.
  • Stabilizers may further include, human serum albumin, bovine serum albumin and gelatin.
  • Tonicifying agents may include sugars and organic salts.
  • Sugars may include, but are not limited to, monosaccharides such as xylose, mannose, glucose and fructose, disaccharides such as trehalose, lactose, maltose and sucrose, trisaccharides such as raffinose, polysaccharides such as dextran, sugar alcohols such as mannitol, sorbitol and glycerol, and cyclitols such as inositol.
  • Amino acids may include but are not limited to glycine, alanine and lysine.
  • Inorganic salts may include but are not limited to sodium chloride, potassium chloride, calcium chloride, dibasic sodium phosphate, monobasic sodium phosphate potassium phpsphate and sodium hydrogencarbonate.
  • Organic salts include but are not limited to sodium citrate, potassium citrate and sodium acetate.
  • Buffering agents may include, but are not limited to, Tris, phosphate bufferand citrate buffer.
  • Surfactants may include, but are not limited to polysorbate 80 and polysorbate 20.
  • Diluents may include, but are not limited to USP Water for Injection or sterile saline such as phosphate buffered saline.
  • Preservatives may include, but are not limited to, benzyl alcohol, chlorobutanol, methylparaben, propylparaben, phenol, m-cresol, benzalkonium chloride, benzethonium chloride, phenoxyethanol, phenylethyl alcohol, chlorhexidine, benzoic acid, phenylmercuric salts and thimerosal. Use in humans
  • PEG-GM-CSF may be used in humans for the same indications as GM- CSF for treatment of patients requiring increased proliferation of white blood cells, often with diseases characterized by neutropenias, aberrant blood cell maturation or reduced leukocyte production.
  • An important clinical application of GM-CSF is treatment of neutropenia following chemotherapy, radiation therapy and bone marrow transplantation. Such therapy can help counter related predispositions to infection and hemorrhage.
  • Indications include:
  • PEG-GM-CSF may be used to enhance tolerance to cytotoxic drugs, such as chemotherapeutic agents, allowing for increased dosing.
  • PEG-GM-CSF may be used to activate resting cancerous cells, making them more susceptible to chemotherapy.
  • PEG-GM-CSF may also be used to treat Crohn's disease, as a vaccine adjuvant (5,679,356), as a treatment for neonatal sepsis, to lower cholesterol levels, as a treatment for radiation exposure, and as a treatment for alveolar proteinosis (6,019,965),
  • PEG-GM-CSF may be used to modulate host defences against infectious disease, including bacterial/ and fungal infections; it may also be used as an adjunctive agent with treatments for fungal and mycobacterial infections.
  • PEG-GM-CSF may be used to combat treatment-related neutropenia, as a prophylaxis and treatment for opportunistic infections, as an enhancer of retroviral treatments, and to reduce HlV expression.
  • PEG-GM-CSF may furthermore be used to modulate host defences for improved anti-tumour or leukemic cell activity.
  • PEG-GM-CSF including oral and mouthwash formulations, may also be used to treat complications of chemotherapy such as mucositis, stomatitis and diarrhea. Wound healing may be promoted by topical or intradermal PEG-GM-CSF.
  • PEG-GM-CSF uses in humans may also be considered to include combinations of PEG-GM-CSF with other growth factors that have synergistic effects including but not limited to G-CSF, IL-6, lL-5, IL-3, MMF, SCF, Meg-CSF, IL-4, erythropoietin, IL-1 , IL-2, interferon-alpha, BCDF and TNF.
  • AAA amino acid analysis
  • AEX anion-exchange chromatography
  • ANC absolute neutrophil count
  • CE capillary electrophoresis
  • HPLC high-performance liquid chromatography
  • MALDI-MS matrix-assisted laser desorption ionization mass spectrometry
  • NIBSC National Institute for Biological Standards and Control
  • PEG poly(ethylene glycol); PK, pharmacokinetic;
  • RP-HPLC reversed-phase high-performance liquid chromatography;
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis;
  • SEC size-exclusion chromatography.
  • Materials Acetonitrile, HPLC grade LeucotropinTM (Cangene's GM-CSF) DL-lysine Mannitol MiIIi-Q water
  • LEUCOTROPINTM (200 mg total) was produced at Cangene Corporation at 2 mg/ml in 10 mM sodium phosphate, 150 mM NaCI pH 7.0. LEUCOTROPINTM had previously been characterized for identity (Western blot), concentration (A 2 so), purity (anion exchange, size- exclusion chromatography), and biological potency (TF-1/MTT bioassay; data not shown). LEUCOTROPINTM was dialyzed using SnakeSkin ® dialysis tubing, 3500 MWCO (Pierce Endogen) in 10 mM sodium phosphate buffer, pH 7.0 for 48 h at 4°C prior to conjugation, with buffer changed twice.
  • SnakeSkin ® dialysis tubing 3500 MWCO (Pierce Endogen) in 10 mM sodium phosphate buffer, pH 7.0 for 48 h at 4°C prior to conjugation, with buffer changed twice.
  • the conjugation reaction was carried out at 4°C with stirring in 10 mM sodium phosphate buffer, pH 7.0 plus 20 mM sodium cyanoborohydride at a 5:1 mol ratio of 20 kDa PEG-butyraldehyde:protein. Reaction progress was monitored by size-exclusion chromatography. The reaction was quenched with an excess of DL-lysine (Sigma) after 18 hr.
  • Reaction product was characterized by capillary electrophoresis (CE) on a Beckman P/ACE System 5010 using a 57 cm, 75 ⁇ m I.D., 375 ⁇ m O. D. eCAPTM silica capillary wound in an eCAPTM capillary cartridge, 100 x 200 urn aperture. Buffer was 100 mM sodium phosphate pH 8.3 run at 8.0 kV; UV absorbance was monitored at 200 nm. Four peaks were observed with CE ( Figure 1), corresponding to non-PEGylated human GM-CSF and human GM-CSF with 1 , 2, or 3 molecules of PEG attached. Purification and Confirmation of Identity of monoPEGylated GM-CSF
  • Reaction product was dialyzed using SnakeSkin ® dialysis tubing, 3500 MWCO (Pierce Endogen) in 20 mM sodium phosphate pH 8.0 for 48 h at 4 0 C, changing buffer twice.
  • the dialyzed PEGylation reaction product was purified on an AKTA Explorer FPLC system equipped with a 20 ml XK 16/10 column packed with Q Sepharose FF resin and a 50 ml Superloop.
  • Elution buffer A was 20 mM sodium phosphate pH 8.0
  • Buffer B was 20 mM sodium phosphate pH 8.0 plus 500 mM NaCI.
  • Sample was eluted using a linear AB gradient from 0 to 225 mM NaCI.
  • Fractions were analyzed by SDS-PAGE. Fractions containing only GM-CSF with a single PEG chain per molecule (;.e., monoPEGylated GM-CSF) were pooled and concentrated in Centricon Plus-20 ultracentrifugal filters (Millipore). A buffer exchange of PEG-GM-CSF was conducted by passing 10 mM sodium phosphate, 40 mM NaCI, 30 mg/ml mannitol pH 7.4 through Centricon Plus-20 centrifugal filter devices. The concentration of protein was obtained by measuring absorbance at 280 nm (A 2 so) and calculated using the molar extinction coefficient of 14180 M "1 cm "1 (derived from the amino acid composition of GM-CSF).
  • Sample was diluted to 0.5 mg protein/ml final concentration with 10 mM sodium phosphate, 40 mM NaCI, 30 mg/ml mannitol pH 7.4.
  • PEG-GM-CSF was subjected to a final 0.2 um sterile filtration using a 25 mm HT Tuffryn ® syringe filter (Pall Scientific), deposited in 1 ml aliquots into 3 ml sterile borosilicate vials (Comar), stoppered, capped and stored at -80 0 C.
  • a sample from the fill lot was tested for endotoxin levels using the limulus amoebocyte lysate assay and was found to contain less than 25 endotoxin units/ml, indicating that samples were sterile and pyrogen-free.
  • Final protein concentration of purified monoPEGylated PEG-GM-CSF was determined by UV absorbance at 280 nm and quantitative amino acid analysis on a Beckman 6300 high performance analyzer using System Gold software. Norleucine was added as an internal standard to all samples analyzed by amino acid analysis, and samples were hydrolyzed in 6 N HCI containing 0.1% phenol for 1 hr at 160 0 C, dried under vacuum and re-dissolved in 200 Dl of running buffer.
  • GM-CSF and PEG-GM-CSF were subjected to trypsin digestion and analysis by reversed-phase HPLC to construct peptide maps, in order to determine the site of monoPEGylation.
  • Either GM-CSF or the PEG-GM-CSF conjugate were buffer exchanged to 1 mg/ml protein concentration in 500 mM Tris buffer, pH 8.1.
  • TPCK-trypsin (0.25 mg/ml in 1 mM HCI solution) (Worthington Enzymes) was added for a final 1 :25 ratio (w/w) of enzyme:protein and incubated at 37 0 C in 0.5 M Tris, pH 8.1.
  • the site of PEG attachment on PEG-GM- CSF was determined using a combination of reversed-phase HPLC analysis and purification, mass spectrometry, and N-terminal peptide sequencing. Trypsin-digested sample was purified by reversed-phase chromatography and fractions corresponding to peptide peaks were collected and dried in a SpeedVac. One peak appeared in the digested PEG-GM-CSF sample that did not appear in the digested GM-CSF sample ( Figure 3).
  • MALDI-MS QStar XL MALDI qTOF (Applied Biosystems) dihydroxybenzoic acid (DHB) matrix
  • DLB dihydroxybenzoic acid
  • the PEGylated peptide peak (obtained from the appropriate RP-HPLC fraction) from the trypsin digest of PEG-GM-CSF was subjected to five cycles of N- terminal Edman sequencing.
  • the obtained amino acid sequence of Pro-Ala-Arg (PAR) in the PEGylated peptide corresponds to residues 2-4 of GM-CSF (data not shown). There are no regions in the sequence of GM-CSF other than at positions 2-4 that contain the PAR sequence, suggesting that PEGylation occurred on the tryptic peptide 1 AIa-PrO-AIa- Arg 4 ( 1 APAR 4 ).
  • the APAR peptide Since the conjugation chemistry employed is specific to primary amino groups at the conditions used, the APAR peptide would only have a single possible site for PEG attachment, at the alpha-amine on the N-terminus. Because the N-terminus of the APAR peptide is also the N-terminus of the full-length GM-CSF molecule, we conclude that, in the monoPEGylated sample obtained, PEGylation occurred substantially at the N- terminus of the full-length GM-CSF protein sequence.
  • Assay media was RPMI-1640 plus 0.001 mg/ml insulin, 0.001 mg/ml transferrin, 0.1 Omol/ml 2-mercaptoethanol, 0.02 mg/ml of gentamicin.
  • TF-1 cells were added to all wells and incubated at a final well concentration of about 2.67X10 5 cells/ml in a 96-well tissue culture plate for 72 hr at 37 DC, 5% CO 2 . After 72 h incubation, one duplicate plate received 50 Dl of 4 mg/ml MTT (a tetrazolium salt) per well followed by incubation for 3 hr at 37 GC, 5% CO 2 .
  • MTT a tetrazolium salt
  • Plates were then centrifuged at 1770 g for 15 minutes, supematants were aspirated and 50 Dl of isopropanol were added to each well. Plates were shaken for 5 minutes and absorbance, which increases with cell proliferation in this assay, was read at 595 nm on a 96-well plate reader. The other duplicate plate was placed on ice to minimize additional cell proliferation, 30 Dl aliquots were removed and added to 30 Dl of Trypan blue stain. Live cells were counted on a hemacytometer to determine live cell concentration from each well.
  • the log dose-response curve for the 1st International Reference Standard was fit using a four-parameter logistic model and estimates of the curve fit parameters obtained. The upper and lower asymptotes, plus the slope factor were then fixed prior to fitting the remaining sample dose-response curves. From the curve fits, estimates of EC50 (the concentration of growth factor that results in 50% maximum response) were obtained. The ratio of the EC50 value obtained for the Reference standard divided by the EC50 value for each Test Sample gives a relative potency that is then multiplied by the assigned potency of the Reference Standard to obtain a potency estimate for each Test Sample.
  • Cangene's PEGylated versions of GM-CSF exhibit significantly enhanced potency (5.4- 6.7 fold) over the non-PEGylated Leucotropin and the 1st International Reference Standard. Similar enhancement was seen in both the MTT bioassay and direct cell counting methods for determining cell proliferation. There was no significant difference in measured potency between the 2OkDa, 3OkDa and 4OkDa PEG-GM-CSF.
  • animals treated with the 3OkDa and 40 kDa PEG-GM-CSF demonstrated an ANC recovery comparable to controls that were treated with supportive care only.
  • the rate of neutrophil loss was slowed to 0 from day 3 to 5 ( Figure 6, squares), relative to negative control monkeys receiving autologous serum ( Figure 6, rectangle).
  • Significant improvement (about 10x increase) in ANC was observed from day 8 to 11 following the second PEG-GM-CSF dosing, on day 7 ( Figure 6).
  • the recovery of animals treated with the 20 kD PEG-GM-CSF is surprising given the concentration of PEG-GM-CSF in the serum.
  • the serum levels of 40 kD PEG-GM-CSF reached about 650,000 pg/ml compared to about 280,000 ⁇ g/ml for the 30 kD PEG-GM-CSF and about 250,000 pg/ml for the 30 kD PEG-GM-CSF ( Figure 7).
  • the serum levels for the 30 kD and 40 kD PEG-GM-CSF molecule appeared to remain at their peak for about 2 to 3 days after the first injection, whereas the levels of 20 kDa PEG-GM-CSF peaked on the day of injection and rapidly decreased.
  • GM-CSF is a key proinflammatory cytokine.
  • reactions at GM-CSF injection sites such as swelling, redness and tenderness, are common side effects.
  • Subcutaneous injection of GM-CSF or 20, 30, or 4OkDa PEG-GM-CSF resulted in localized inflammation in a primate model of myelosuppression.
  • the 2OkDa PEG-GM-CSF produced mild inflammation that was easily treatable with antihistamine (diphenhydramine), the 30 and 40 kDa PEG-GM-CSF molecules caused inflammation that was more severe.
  • the unexpected finding suggests that 2OkDa PEG- GM-CSF is the optimal choice of PEGylated GM-CSF.

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Abstract

L'invention concerne un polypeptide du GM-CSF humain pleine longueur, non glycosylé, qui comporte une seule molécule de polyéthylène glycol fixée de manière covalente par l'intermédiaire d'un acide aminé N-terminal. Selon l'invention, ce polypeptide du GM-CSF humain auquel est fixée la molécule de polyéthylène glycol présente, de façon inattendue, une capacité accrue de stimulation de la réplication de cellules en culture par rapport à la molécule de GM-CSF non modifiée. Cette capacité accrue de stimuler la division cellulaire en culture se manifeste par l'activité accrue des neutrophiles dans un modèle animal.
PCT/CA2006/000885 2005-06-02 2006-06-02 Gm-csf humain modifie au poly(ethylene glycol) qui presente une activite biologique accrue WO2007009208A1 (fr)

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WO2010030366A2 (fr) * 2008-09-11 2010-03-18 Nektar Therapeutics Réactifs polymères alpha-hydroxy aldéhydiques et cétoniques et procédé de conjugaison
CN106841419A (zh) * 2016-12-29 2017-06-13 合肥安德生制药有限公司 一种聚肽的快速检测方法
WO2017214249A3 (fr) * 2016-06-08 2018-01-18 Janssen Biotech, Inc. Variants de gm-csf et procédés d'utilisation
WO2021185921A1 (fr) * 2020-03-17 2021-09-23 Drugrecure Aps Formulation liquide de gm-csf pour inhalation
WO2021222806A1 (fr) * 2020-05-01 2021-11-04 Icahn School Of Medicine At Mount Sinai Compositions et méthodes de prévention, de détection et de traitement de la maladie intestinale inflammatoire
WO2023055378A1 (fr) * 2021-09-30 2023-04-06 Savara Inc. Méthodes de traitement de la protéinose alvéolaire pulmonaire auto-immune

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010030366A3 (fr) * 2008-09-11 2011-09-01 Nektar Therapeutics Réactifs polymères alpha-hydroxy aldéhydiques et cétoniques et procédé de conjugaison
US8492503B2 (en) 2008-09-11 2013-07-23 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US9228053B2 (en) 2008-09-11 2016-01-05 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US9579392B2 (en) 2008-09-11 2017-02-28 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US11634540B2 (en) 2008-09-11 2023-04-25 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US9908970B2 (en) 2008-09-11 2018-03-06 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
WO2010030366A2 (fr) * 2008-09-11 2010-03-18 Nektar Therapeutics Réactifs polymères alpha-hydroxy aldéhydiques et cétoniques et procédé de conjugaison
US10759906B2 (en) 2008-09-11 2020-09-01 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US11220575B2 (en) 2008-09-11 2022-01-11 Nektar Therapeutics Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
US11208450B2 (en) 2016-06-08 2021-12-28 Janssen Biotech, Inc. GM-CSF variants and methods of use
WO2017214249A3 (fr) * 2016-06-08 2018-01-18 Janssen Biotech, Inc. Variants de gm-csf et procédés d'utilisation
US10562948B2 (en) 2016-06-08 2020-02-18 Janssen Biotech, Inc. GM-CSF variants and methods of use
CN106841419B (zh) * 2016-12-29 2019-08-13 合肥安德生制药有限公司 一种聚肽的快速检测方法
CN106841419A (zh) * 2016-12-29 2017-06-13 合肥安德生制药有限公司 一种聚肽的快速检测方法
WO2021185921A1 (fr) * 2020-03-17 2021-09-23 Drugrecure Aps Formulation liquide de gm-csf pour inhalation
CN115297844A (zh) * 2020-03-17 2022-11-04 德拉格雷丘尔公司 用于吸入的gm-csf的液体制剂
WO2021222806A1 (fr) * 2020-05-01 2021-11-04 Icahn School Of Medicine At Mount Sinai Compositions et méthodes de prévention, de détection et de traitement de la maladie intestinale inflammatoire
WO2023055378A1 (fr) * 2021-09-30 2023-04-06 Savara Inc. Méthodes de traitement de la protéinose alvéolaire pulmonaire auto-immune

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