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WO2003089468A1 - Methodes et moyens de production de proteines avec des modifications post-traductionnelles preetablies - Google Patents

Methodes et moyens de production de proteines avec des modifications post-traductionnelles preetablies Download PDF

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Publication number
WO2003089468A1
WO2003089468A1 PCT/NL2002/000257 NL0200257W WO03089468A1 WO 2003089468 A1 WO2003089468 A1 WO 2003089468A1 NL 0200257 W NL0200257 W NL 0200257W WO 03089468 A1 WO03089468 A1 WO 03089468A1
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Prior art keywords
erythropoietin
molecule
sialic acid
molecules
acid residues
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PCT/NL2002/000257
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English (en)
Inventor
Dirk Jan Elbertus Opstelten
Johan Christiaan Kapteyn
Petrus Christianus Johannes Josephus Passier
Ronald Hendrik Peter Brus
Abraham Bout
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Janssen Vaccines and Prevention BV
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Crucell Holand BV
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Priority to AU2002307635A priority Critical patent/AU2002307635A1/en
Priority to PCT/NL2002/000257 priority patent/WO2003089468A1/fr
Priority to EA200602163A priority patent/EA012340B1/ru
Priority to CNB028216903A priority patent/CN100347306C/zh
Priority to AT02770322T priority patent/ATE542904T1/de
Priority to PCT/NL2002/000686 priority patent/WO2003038100A1/fr
Priority to KR1020067008079A priority patent/KR100602772B1/ko
Priority to EA200400605A priority patent/EA008220B1/ru
Priority to EP02770322A priority patent/EP1440157B1/fr
Priority to ES02770322T priority patent/ES2381104T3/es
Priority to KR1020067008077A priority patent/KR100737639B1/ko
Priority to US10/494,140 priority patent/US7304031B2/en
Priority to IL16167402A priority patent/IL161674A0/xx
Priority to DK02770322.2T priority patent/DK1440157T3/da
Priority to CN2007101494988A priority patent/CN101177700B/zh
Priority to MXPA04003940A priority patent/MXPA04003940A/es
Priority to CA2465007A priority patent/CA2465007C/fr
Priority to EP10177590.6A priority patent/EP2292770B1/fr
Priority to AU2002335585A priority patent/AU2002335585B2/en
Priority to BR0213402-0A priority patent/BR0213402A/pt
Priority to KR1020047006311A priority patent/KR100692784B1/ko
Priority to JP2003540365A priority patent/JP4583029B2/ja
Priority to CA2756610A priority patent/CA2756610C/fr
Publication of WO2003089468A1 publication Critical patent/WO2003089468A1/fr
Priority to IL161674A priority patent/IL161674A/en
Priority to NO20042209A priority patent/NO20042209L/no
Anticipated expiration legal-status Critical
Priority to US11/102,073 priority patent/US7297680B2/en
Priority to US11/657,202 priority patent/US7785833B2/en
Priority to US11/888,776 priority patent/US7696157B2/en
Priority to IL201673A priority patent/IL201673A/en
Priority to IL201674A priority patent/IL201674A/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the field of medicine.
  • the invention further relates to the production of proteins . More particularly the present invention relates to the production of recombinant proteins for use as a therapeutically active constituent of a pharmaceutical preparation.
  • Recombinant expression systems for the production of proteins are widely known.
  • human recombinant proteins are manufactured with the use of a cellular expression system. These systems range from bacteria, yeast and fungi to plant cells, and from insect cells to mammalian cells. Most of these cell-based systems are only suited for a specific class of proteins.
  • Consideration for the production host and expression system of choice generally relate to ease of use, cost of culturing, growth characteristics, production levels and the ability to grow on serum-free medium.
  • Bacterial systems as well as yeast systems are in many aspects the system of choice, since the cost of culturing is low and they are in general easy to use in comparison to other, higher eukaryotic systems.
  • prokaryotic- and lower eukaryotic systems differ in a number of aspects from higher eukaryotic systems (such as mammalian cells).
  • higher eukaryotic systems such as mammalian cells
  • post-translational modifications like glycosylation do no occur in bacterial systems, while many mammalian proteins depend on these post-translational modifications to be therapeutically active.
  • the cellular expression systems mentioned above might also differ in the capacity to exert other co- and post-translation modifications such as folding, phosphorylation, ⁇ - carboxylation, and ⁇ -hydroxylation.
  • the co- and post-translational modifications are cell- and species specific and depend on the expression system being applied.
  • CHO and BHK cells are frequently the expression system of choice when a mammalian expression system is required. These mammalian cells are able to produce proteins efficiently and allow generally for proper processing and folding. More recently, a human cell line was proposed for production of human proteins. The human post-translational modifications of human proteins are thought to have a positive effect on the pharmacokinetic and/or pharmacodynamic properties in general (WO 00/63403) .
  • Disorders affecting the CNS encompass different kinds of afflictions such as acute brain damage, neurodegenerative diseases and other dysfunctions such as epilepsy, schizophrenia and mood disorders. With the increase of the number of elderly people in the worldwide population in the next decades, an increasing number of people become at risk of degenerative neurological diseases related to the CNS. Two main examples of such disorders are Parkinson's and Alzheimer's Disease. Other pathological disorders that might afflict neural cells and tissues are due to injuries that might be a result of hypoxia, seizure disorders, neurotoxin poisoning, multiple sclerosis, hypotension, cardiac arrest, radiation or hypoglycemia.
  • Neural injuries might also occur during surgical procedures such as aneurysm repair or tumor resection.
  • the treatment of the CNS disorders has been mainly via administering pharmaceutical compounds.
  • the main drawback that has been appreciated in the art is the limited ability to transport drugs across the blood-brain barrier and the drug tolerance that is acquired by patients to whom these drugs are administered for prolonged periods of time.
  • Other treatments for degenerative diseases related to the CNS include neurological tissue grafting: the use of fetal cells for neuro-transplantation has been explored. A clear improvement in the condition of Parkinson' s patients was reported by several groups after applying such techniques (Freed et al. 1992; Widner et al. 1992) .
  • fetal tissue is most likely never a homogeneous cell population, so it is not a well-defined source of cells, and the question remains whether there will be a sufficient and adequate constant supply of fetal tissue for these purposes.
  • Another method makes use of the fact that in the adult mammalian neural tissue multipotent neural stem cells exist, that are capable of producing progeny that differentiate into neurons and glia (Reynolds and Weiss 1992) . Methods have been provided for the proliferation of these stem cells to provide large numbers of neural cells that can differentiate into neurons and glia using various growth factors (U.S. Pat. No.
  • EPO Erythropoietin
  • EPO-R EPO-receptor
  • EPO a protein famous for its role in differentiating hematopoietic stem cells into red blood cells, also seems to increase the number of neural progeny that are generated from proliferated neural stem cells.
  • Several methods of inducing the differentiation of multipotent neural cells into neurons and methods of treating neurodegenerative diseases or acute brain injuries by producing neurons from such cells using EPO are described in U.S. Pat. No. 6,165,783.
  • EPO not only has a hematopoietic effect, but that there also might be a role for EPO in neural tissues.
  • non-erythroid cells express the EPO-R and respond to recombinant EPO in vi tro and in vivo.
  • neural cell lines like NT2, PC12 and SN6.10.2.2. express the EPO-R, and exposure of PC12 cells to recombinant human EPO causes a rapid influx of calcium from outside the cells and increases the intracellular concentration of monoamines (Masuda et al. 1993) .
  • Recombinant human EPO was also found to augment choline acetyltransferase (ChAT) activity in primary cultured mouse septal neurons and in the cholinergic hybridoma cell line, SN6.10.2.2. (Konishi et al. 1993). In the developing and mature brain of rodents, monkeys and humans expression of EPO and the EPO-R has also been detected, (Marti et al.
  • EPO and EPO-R have been localized to neurons and glia cells in spinal cord and brain during fetal human development (Juul et al . 1998). The distribution of EPO and EPO-R proteins in fetal and adult brain has been determined by immunohistochemistry
  • EPO In the human fetus EPO is expressed at a level that is comparable to that in the liver and kidneys, which are the most relevant sites for circulating EPO at this stage of development ( Indianapolis et al . 2001). Also in mice, it has been shown that there is a high constitutive level of EPO mRNA in the brain compared to the kidneys and the liver (Marti et al. 1996) . These observations suggested a role of EPO in the development of the CNS (Dame et al. 2001). EPO protein has also been detected in the cerebrospinal fluid (CSF) of human neonates and adults (Juul et al . 1997; Buemi et al. 2000).
  • CSF cerebrospinal fluid
  • EPO in CSF The concentration of EPO in CSF is relatively high in neonates and decreases to lower but nevertheless detectable levels in adults (Juul et al. 1997; Marti et al . 1997). Compared to healthy individuals, patients with old cerebrovascular disease and patients with depression have an elevated level of EPO in the CSF (Naka ura et al. 1998). That EPO is produced locally in the brain is strengthened by the observation that EPO does normally not cross the intact blood-brain barrier because there is no correlation between the concentration of EPO in the serum and in the CSF (Marti et al . 1997) .
  • the expression of the EPO gene in the kidneys as well as in the brain is increased under hypoxic conditions.
  • the expression is regulated by the transcription factor hypoxia-inducible factor-1, which is activated by a variety of stress signals, including hypoxia. It has recently been demonstrated in mice that the EPO mRNA levels markedly increase within 4 hours upon the exposure to hypoxia (Chikuma et al . 2000). Yet, the EPO mRNA levels in the kidneys decreased within 8 hours during continuous hypoxia whereas the EPO mRNA levels in the cerebrum remained the same.
  • EPO heamatopoietic function
  • the expression of EPO in monkey- and mouse brain has been shown to be 3 to 20-fold increased respectively, under hypoxic conditions (Digicaylioglu et al. 1995; Marti et al . 1996). Hypoxic effects on the expression of EPO have also been demonstrated in vitro .
  • hypoxia caused a more than 100-fold up-regulation of the mRNA levels of EPO (Marti et al. 1996). Furthermore, it has been found that the mRNA level of the EPO-R is also upregulated under hypoxic conditions in vivo . This was found in studies in which the middle cerebral artery of rats was occluded and in which the increase of EPO-R mRNA was detected by in situ hybridization in the periphery of a cerebrocortical infarct (Sadamoto et al. 1998). It suggests that neurons increase their sensitivity to EPO by increasing their number of EPO-R under hypoxic and/or ischemic conditions. Finally, it has recently been confirmed by immunohistochemical methods that the expression of immuno-reactive EPO and EPO-R is also upregulated in fresh infarcts inside the human ischemic/ hypoxic brain (Siren et al . 2001).
  • EPO can act as a neurotrophic factor.
  • Neurotrophic factors are defined as humoral molecules acting on neurons to influence their development, differentiation, maintenance, and regeneration (Konishi et al. 1993) .
  • Neurotrophic effects of EPO have first been shown in vi tro, in cultured neurons.
  • recombinant human EPO in a dose-dependent manner, protects cultured embryonic rat hippocampal and cerebral cortical neurons from glutamate toxity (Morishita et al. 1997).
  • Hypoxia-induced cell death in cultures of postnatal rat hippocampal neurons also has been shown to be reduced by EPO (Lewczuk et al. 2000).
  • recombinant mouse EPO protects cultured rat cortical neurons, but not astroglia from glucose deprivation-induced hypoxia and from the neurotoxic effects of ( ⁇ ) - ⁇ -amino-3-hydroxy-5- methylisoxazole-4-propionic acid (Sinor and Greenberg 2000). Furthermore, recombinant human EPO has recently been proven not only to protect neural cell cultures form hypoxia but also from serum deprivation or kainic acid exposure (Siren et al. 2001). The neurothropic function of EPO has also been shown in rats, which were treated with soluble EPO-R that competed with the natural EPO-R for binding endogenous EPO.
  • EPO and its receptor are identified in endothelial cell in vitro and in vivo (Anagnostou et al. 1994). Jn vitro experiments demonstrated that recombinant human EPO stimulates cell migration and proliferation in endothelial cell cultures. These in vitro results were further extended by the effect of human recombinant EPO on the stimulation of new blood vessel formation in the chick chorioallantoic membrane (Ribatti et al . 1999) . In accordance in mice, EPO was found to play an important role in the 17-beta-estradiol dependent angiogenesis in the uterine endometrium (Yasuda et al . 1998) .
  • EPO Endothelial growth factor
  • myoblast C2C12 cell-line EPO enhanced the proliferation and reduced the differentiation and fusion into myotubes of these cells in vi tro (Ogilvie et al. 2000).
  • the receptor of EPO could also be detected on primary satellite cells isolated from skeletal muscle from mice and on C2C12 cells at the mRNA and at the protein level.
  • EPO has been shown to stimulate DNA-synthesis and proto-oncogene expression in rat vascular smooth muscle cells.
  • EPO embryogenesis EPO is expressed in the heart and mice lacking EPO or EPO-R expression display cardiac defects, demonstrated by ventricular hypoplasia and a reduction in the number of proliferating cardiomyocytes and increased apoptosis (Wu et al. 1999). Yu et al . (2001) demonstrated that the cardiac phenotype in the EPO-R knockouts could be rescued by crossing these mice with transgenic mice, harboring the human EPO receptor gene. In addition to the restoration of erythropoiesis, the cardiac defect was corrected and apoptosis was markedly reduced. Furthermore, also apoptosis in liver and brain was significantly reduced in these mice.
  • CHF congestive heart failure
  • selectins A certain family of glycoproteins, named selectins, play an important role in the initial steps of adhesion of leukocytes to the endothelium in ischemia/reperfusion injury. There are three members in the selectin family: P- selectin, E-selectin and L-selectin. L-selectin is constitutively expressed on leukocytes, whereas P-selectin and E-selectin are found on the membrane upon activation. P-selectin is present in Weibel-Palade bodies of endothelial cells and alpha-granules of platelets.
  • P-selectin is translocated to the cell surface, within 10-20 minutes (Lorant et al. 1991; Weyrich et al . 1995) , whereas E-selectin is expressed on endothelial cells after de novo synthesis, which takes approximately 4-6 hours (Bevilacqua et al . 1989).
  • Selectins initiate the rolling of the leukocytes along the endothelium. P-selectin is the most important selectin for this first step of leukocyte rolling (Lefer and Lefer 1996) .
  • the rolling of the leukocytes reduces the velocity of the leukocytes in the bloodstream and allows a more firm interaction between the leukocytes and the endothelium by other adhesion molecules (integrins) .
  • Firm adhesion is followed by transendothelial migration.
  • Infiltration of the neutrophils can be observed 3 hours following reperfusion. After this time period, reperfusion injury with its resulting cell death takes place (Armstead et al . 1997).
  • P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinty ligand for P-selectin and to a lesser extent for L-selectin and E-selectin (Moore et al. 1994).
  • the oligosaccharides in PSGL-1 are recognized by the lectin domain of the selectins.
  • the highly branched N-linked oligosaccharides are thought to increase the volume of the protein such that it is not easily filtered by the kidney (Takeuchi et al. 1989; Misaizu et al . 1995).
  • the ulti- antennary sugar structures may also have a role in targeting of EPO to the bone marrow where erythropoiesis occurs (Takeuchi et al . 1989).
  • the role of the O-linked oligosaccharides present on EPO is relatively unclear and some data have suggested that there is only a limited role for the O-linked sugar in EPO present in circulation and that has an effect on he atopoiesis .
  • human neural glycoproteins are characterized by their glycosylation, which has been referred to in literature as 'brain-type' glycosylation (Margolis and Margolis 1989; Hoffmann et al . 1994).
  • serum-type' glycosylated proteins i.e., glycoproteins circulating in the blood
  • brain-type glycosylated proteins characteristically possess complex- type N-linked sugars that are modified with ⁇ l,3-linked fucose attached to N-acetyl-glucosamine in lactosamine-type antennae thereby forming Lewis x or sialyl-Lewis x structures (Fig. 5) .
  • Lewis x structures There are two types of Lewis x structures: One with a terminal galactose residue and one with a terminal N-acetyl-galactosamine (GalNac) residue. If these terminal groups are linked to a sialic acid, the Lewis x structure is called a sialyl Lewis x structure.
  • Another difference between serum-type and brain-type oligosaccharides is that the latter often contain terminal N-acetyl-glucosamine and/or terminal galactose, and may include a terminal N-acetyl-galactosamine modification, whereas serum-type oligosaccharides usually contain only low amounts of such structures. It has also been suggested that brain-type N-oligosaccharides characteristically contain high amounts of bisecting N-acetyl-glucosamine (Hoffmann et al. 1994).
  • O-glycan structure is in brain-type EPO, but the limited role for this oligosaccharide in serum might imply that there is a more important role for this type of glycosylation in brain-type EPO. It is very likely as well, that in accordance with the N-glycans, also the O-glycan has a differential glycosylation pattern between serum-type and brain-type. Oligosaccharides that are generally found on proteins circulating in the serum contain often heavily galactosylated structures. This means that a galactose is linked to a peripheral N-acetyl-glucosamine thereby forming a lactosamine structure.
  • the glycoprotein is in this way protected from endocytosis by the N-acetyl-glucosamine receptors (i.e., receptors that recognize terminal N- acetyl-glucosamine) present in hepatic reticuloendothelial cells and macrophages (Anchord et al . 1978; Stahl et al. 1978).
  • Serum-type oligosaccharides usually also contain terminal sialic acids (also often referred to as neuraminic acid) which protect the glycoprotein from clearance through the asialoglycoprotein receptor. These clearance mechanisms specifically apply to glycoproteins circulating in the blood and are probably lacking in the human central nervous system (CNS) (Hoffmann et al . 1994).
  • Transferrin is a protein capable of interacting with iron via two iron-binding sites in the protein. Iron uptake by cells occurs through receptor-mediated endocytosis of the transferrin protein that is bound to iron.
  • Human serum-type transferrin carries two N- glycosylation sites (Asn413 and Asn ⁇ ll) that are generally occupied by disialylated, bi-antennary oligosaccharide chains. Most transferrin proteins seem to have 4 sialic acid residues (i.e.
  • Serum-type transferrin does not contain polylactosamines or fucose residues. A minor amount of serum-type transferrin carries sialylated tri-antennary oligosaccharides. It has been found that rarely also tetra- antennary structures occur.
  • glycosylation of transferrin is used as a marker for carbohydrate-deficient glycoprotein syndromes (CDGS) .
  • CDGS carbohydrate-deficient glycoprotein syndromes
  • EPO EPO
  • serum-type EPO or a 'renal-type' , or a 'urinary-type' EPO
  • EPO EPO
  • EPO protein that was produced by cultured rat brain cells was found to be significantly smaller than the EPO protein present in circulation. This mass difference might be the reason for the different biological roles in the brain and in circulation. It was found, that although brain-type EPO produced on these cultured rat brains is approximately 15% smaller than serum-type EPO (presumably mainly due to differences in sialylation) , this brain-type EPO is more active in vitro in erythroid colony stimulation at low ligand concentrations (Masuda et al . 1994).
  • sialyl Lewis x structures are expressed on leukocytes and are rapidly expressed on vascular endothelial cells and cardiac myocytes following myocardial ischemia/reperfusion injury in vivo (Yamazaki et al. 1993). Furthermore, sialyl Lewis x structures are also induced on the surface of endothelial cells and cardiomyocytes by hypoxia/reoxygenation in vitro (Seko et al. 1996).
  • sialyl Lewis x oligosaccharide Sle x -OS was shown to be cardioprotective in a feline model of ischemia/reperfusion by reducing cardiac necrosis by 83% (Buerke et al. 1993). In addition reduced adhesion to the endothelium was observed in this model using Sle x -OS. Furthermore, in a similar ischemia/reperfusion model, treatment with Sle x -OS also resulted in a 100% recovery in cardiac function, compared to 71% recovery of cardiac function with saline. In dogs subjected to myocardial infarction/reperfusion, treatment with Sle x -OS demonstrated cardioprotection by a 55% reduction in infarct size (Flynn et al. 1996) .
  • the system of choice thus far has been a production platform on CHO cells from which the higher sialylated EPO forms were purified and used to prepare a medicament for the treatment of patients that suffer from the disorders resulting from a low-red blood cell level.
  • Other cells that were used for these purposes were BHK cells.
  • CHO and BHK cells are capable of generating the correct glycosylation (sialylation) patterns on the recombinant product to yield positive effects in human renal-failure patients.
  • CHO produced EPO does not have the characteristic features of an EPO molecule that is active in the brain or in tissues that involve selectin-based transport.
  • urinary- or serum-type EPO produced on cells such as CHO or BHK
  • urinary- or serum-type EPO is relatively useless in the treatment of disorders related to the Central- or Peripheral Nervous system as well as in the treatment of afflictions related to ischemia/reperfusion induced disorders.
  • This because of its glycosylation pattern that is not suited for these kind of tissues, and also because it renders side effects such as an increase in the number of red blood cells (erythropoiesis) due to its strong hematopoietic activity.
  • No proper production platforms are present in the art that are able to produce significant amounts of recombinant proteins harboring tissue-specific predetermined post- translational modifications such as a brain-type glycosylation on recombinant EPO, and that can be used in the manufacturing of medicaments for the treatment of patients suffering from disorders that require such proteins, as well as the treatment of patients at risk of developing such disorders.
  • Table I Overview of the marker proteins that can be used to characterize cells.
  • Table II Positive control tissues that can be used for some of the marker proteins depicted in Table I.
  • Table III Detailed information (Supplier and Catalogue numbers) of antibodies directed to marker proteins that were used to characterize the PER.C6TM cell line.
  • Table V Monosaccharide composition of the N-linked sugars of PER.C6-EPO and Eprex.
  • FIG. 7 Infarct volumes in untreated rats (control) and Eprex and PER.C6-EPO treated rats based on the ADC maps (Fig. 7A) and the T2 maps (Fig. 7B) generated at 24 h after the onset of reperfusion, using MRI .
  • the present invention provides methods for identifying, selecting and obtaining mammalian cells that are capable of producing proteinaceous molecules, such as peptides and proteins comprising post-translational modifications, wherein said post-translational modifications are predetermined and brought about by the mammalian cell in which the proteinaceous molecule is expressed.
  • the invention further provides methods for obtaining and producing proteinaceous molecules, such as erythropoietin (EPO) , using mammalian cells obtainable according to methods of the present invention and on mammalian cells that have been obtained on the basis of their ability to produce proteins and/or post-translational modifications that are elusive for the predetermined post- translational modification that is desired.
  • EPO erythropoietin
  • the present invention provides mammalian cells that have neural characteristics and properties such that significant amounts of recombinant proteins can be produced that harbor 'neural- or brain- type' properties.
  • the production of recombinant proteins, like brain-type EPO, carrying specific predetermined post- translational modifications, is now feasible by using the methods and means of the present invention.
  • the present invention furthermore provides methods for purifying proteinaceous molecules, wherein said proteinaceous molecules are purified from cell culture on the basis of the predetermined post-translational modification present on the molecule, said predetermined post-translational modification being brought about by the mammalian cell on which the molecule was produced.
  • the present invention furthermore provides for use of a composition of erythropoietin-like molecules selected from the group consisting of one or more muteins of erythropoietin, one or more derivatives of erythropoietin, or a collection of one or more fractions of erythropoietin molecules sialylated to a varying degree, for the preparation of a medicament for the treatment of a disorder selected from the group consisting of ischemia, a reperfusion injury, a hypoxia-induced disorder, an inflammatory disease, a neurodegenerative disorder, and acute damage to the central- or peripheral nervous system, wherein said composition of erythropoietin-like molecules has on a protein content basis a lower erythropoietic activity in vivo than epoetin alfa.
  • the present invention also provides pharmeceutical compositions comprising such erythropoietin-like molecules.
  • the invention also provides methods for
  • the present invention provides a method for identifying a mammalian cell capable of producing a proteinaceous molecule comprising a predetermined post- translational modification, said method comprising the steps of: a) analyzing the post-translational modification on a protein produced by said mammalian cell; and b) determining whether said protein comprises said predetermined post-translational modification.
  • the invention provides a method for selecting a mammalian cell capable of producing a proteinaceous molecule comprising a predetermined post- translational modification, said method comprising the steps of: a) analyzing the presence or absence of a tissue specific marker or a combination of tissue specific markers in said mammalian cell or on the cell surface of said mammalian cell, which marker or combination of said markers is indicative for said predetermined post-translational modification to be present on said proteinaceous molecule; and b) selecting said mammalian cell on the basis of the presence or absence of said tissue specific markers.
  • the invention provides a method for obtaining a mammalian cell from a heterogeneous cell population, said mammalian cell being capable of producing a proteinaceous molecule comprising a predetermined post-translational modification, said method comprising the steps of: a) sorting cells on the basis of the post-translational modifications on proteins produced by said cells in said heterogeneous cell population; and b) selecting the cells capable of producing proteins comprising said predetermined post-translational modification.
  • the invention provides a method for identifying a mammalian cell capable of producing a proteinaceous molecule comprising a predetermined post- translational modification, said method comprising the steps of: providing said mammalian cell with a nucleic acid encoding a protein capable of comprising post-translational modifications, in such a way that said mammalian cell harbors said nucleic acid in an expressible form; culturing said mammalian cell under conditions conducive to the production of said protein; analyzing the post- translational modification on said protein produced by said mammalian cell; and determining whether said post- translational modification present on said protein comprises said predetermined post-translational modification.
  • the invention provides a method for identifying a mammalian cell capable of producing a proteinaceous molecule comprising a predetermined post-translational modification, said method comprising the steps of: providing said mammalian cell with a nucleic acid encoding said proteinaceous molecule capable of comprising post- translational modifications, in such a way that said mammalian cell harbors said nucleic acid in an expressible form; culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule; analyzing the post-translational modification on said proteinaceous molecule produced by said mammalian cell; and determining whether said post-translational modification present on said proteinaceous molecule comprises said predetermined post-translational modification.
  • a proteinaceous molecule as used herein refers to, but is not limited to, molecules such as peptides, polypeptides and proteins, as well as to mutants of peptides, polypeptides and proteins (molecules comprising deletions, pointmutations, swaps and/or chemically induced alterations). It also refers to peptides, polypeptides and proteins carrying tags and/or other proteinaceous and non- proteinaceous labels (e.g., radio-active compounds).
  • An example of such a protein is human EPO, which has besides the renal- or serum-type form, other phenotypes such as a brain-type form.
  • classes of proteins that have certain characteristics that possibly play an important role in the functionality of the protein in certain tissues and that should (when recombinantly expressed) harbor the predetermined post-translational modifications for a proper function are monoclonal antibodies, neurotrophins, cytokines, insulin-like growth factors, TGF- ⁇ like growth factors, fibroblast growth factors, epidermal growth factors, heparin binding growth factors, tyrsosine kinase receptor ligands and other trophic factors. Most of these factors are associated with disease syndromes, and therefore most of the proteins might be used in recombinant form in the treatment of humans, provided that the proteins harbor the post-translational modifications necessary to be active in vivo .
  • proteins should therefore be produced on expression systems that are capable of providing the required post- translational modifications.
  • Examples of such proteins are, but are not limited to, transferrin, Nerve Growth Factor (NGF) , Brain-derived neurotrophic factor, Neurotrophin-3, - 4/5 and -6, Ciliary neurotrophic factor, Leukemia inhibitory factor, Cardiotrophin-1, Oncostatin-M, several Interleukins, GM-CSF, G-CSF, IGF-1 and -2, TGF- ⁇ , Glial- derived neurotrophic factor, Neurturin, Persephin, Myostatin, Fibroblast Growth Factor-1, -2 and -5, Amphiregulin, Acetylcholine receptor inducing activity, Netrin-1 and -2, Neuregulin-2 and -3, Pleiotrophin, Midkine, Stem Cell Factor (SCF), Agrin, CSF-1, PDGF and Saposin C.
  • NGF Nerve Growth Factor
  • SCF Stem Cell
  • Monoclonal antibodies refer to human and humanized antibodies, to parts thereof, and to equivalents such as single chain Fv (scFv) fragments, Fab fragments, CDR regions, variable regions, light chains and heavy chains.
  • Antibodies may be bispecific, trispecific, and so forth; either in naked form or conjugated to antigenic moieties, toxins, fluorescent markers, radiolabels, and the like.
  • a post-translational modification as used herein refers to any modification that is present on or in said proteinaceous molecule. It refers to modifications that are introduced during or subsequent to the translation of said molecule from RNA in vivo or in vitro. Such modifications include, but are not limited to, glycosylation, folding, phosphorylation, ⁇ -carboxylation, ⁇ -hydroxylation, multimerization, sulphide bridging and for instance processing events such as the clipping-off or the addition of one or more amino acids.
  • a predetermined post- translational modification as used herein refers to any post-translational modification that is useful for the selected treatment.
  • predetermined post-translational modification refers to a form of modification that makes the modified protein particularly useful to treat disorders of specific tissues, organs, compartments and/or cells of a human or animal body.
  • the proteinaceous molecule carrying such predetermined post-translational modification is less active in a tissue, organ, compartment and/or cell wherein action is less desired.
  • the proteinaceous molecule carrying such predetermined post- translational modifications does not exert any significant effect (such as detrimental- or other undesired side- effects) other than the tissue, organ, compartment and/or cell that is to be treated.
  • the predetermined post-translational modification causes the protein comprising the predetermined post-translational modification to be cleared from the blood more rapidly, e.g., to reduce adverse side effects.
  • the predetermined post-translational modification can be fully understood in detail in advance, but can also be generally referred to as being a desired state that is required for a proper and wanted activity of the proteinaceous molecule comprising such predetermined post-translational modification, meaning that the detailed modifications present on the proteinaceous molecule of interest might not be fully understood and/or defined, but nevertheless hold activity features that are desired.
  • glycosylation modifications in 0- and/or N-glycans that might be present on a proteinaceous molecule, that might, but do not have to be necessarily desired, are exemplified by structures such as Lewis x, sialyl Lewis x, GalNac, GlcNac, LacdiNAc, ⁇ l,3- linked fucose attached to N-acetyl-glucosamine, terminal N- acetyl-glucosamine, terminal galactose, bisecting N-acetyl- glucosamine, sulphate group and sialic acid.
  • structures such as Lewis x, sialyl Lewis x, GalNac, GlcNac, LacdiNAc, ⁇ l,3- linked fucose attached to N-acetyl-glucosamine, terminal N- acetyl-glucosamine, terminal galactose, bisecting N-acetyl- glucosamine,
  • the mammalian cells of the present invention are preferably human, for the production of human proteins to produce proteins that most likely carry mammalian-, and preferably human, characteristics.
  • human proteins to produce proteins that most likely carry mammalian-, and preferably human, characteristics.
  • neural characteristics such as protein markers that are indicative for neural cells. This does not exclude that a non-neural cell might be extremely useful in producing proteins comprising neural-type post-translational modifications. It depends on the protein activity that is required, to select, identify or obtain a cell that is capable of producing such post-translational modifications.
  • cell-type specific marker proteins in or on a cell does also not exclude the possibility that a certain type of cell is capable of producing a protein carrying the desired post-translational modifications, although this cell expresses some unrelated protein markers. It depends on the selection criteria, as well as on the method for selecting (determining the protein markers and/or the post-translational modifications present on proteins produced in the cell) whether a certain cell is suitable for producing the proteinaceous molecule of interest .
  • the mammalian cells of the invention are immortalized. Immortalization can be brought about in many ways . Examples of methods to obtain immortalized cells are actively transforming a resting cell into a dividing cell by the addition of nucleic acids encoding transforming and/or immortalizing proteins, or through chemical treatment through which endogenous proteins might become transforming, or by taking cells from tumor material.
  • One preferred method to immortalize non- tumorous cells is by the addition of the El region of adenovirus as was shown for cell lines such as HEK293, 911 and PER.C6.
  • HPV Human Papillomavirus
  • El from adenovirus
  • ElA of adenovirus might induce the expression of certain endogenous proteins that have a role in the addition of post-translational modifications on the recombinant proteins to be produced.
  • the present invention provides a method for producing a proteinaceous molecule comprising a predetermined post- translational modification, comprising the steps of: providing a mammalian cell obtainable by methods according to the invention, with a nucleic acid encoding said proteinaceous molecule in such a way that said mammalian cell harbors said nucleic acid in an expressible form; and culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule.
  • the invention provides a method for producing a proteinaceous molecule comprising a predetermined post-translational modification, comprising the steps of: identifying a mammalian cell having the ability to provide the proteinaceous molecule with said predetermined post-translational modification; providing said mammalian cell with a nucleic acid encoding said proteinaceous molecule in such a way that said mammalian cell harbors said nucleic acid in an expressible form; and culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule.
  • the invention provides a method for producing a proteinaceous molecule comprising a predetermined post-translational modification, said method comprising the steps of: identifying a mammalian cell having the ability to provide said proteinaceous molecule with said predetermined post-translational modification; providing said mammalian cell with a nucleic acid encoding said proteinaceous molecule in such a way that said mammalian cell harbors said nucleic acid in an expressible form; culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule; analyzing said post-translational modifications on said proteinaceous molecule so produced; and determining whether said post-translational modification present on said proteinaceous molecule comprises said predetermined post- translational modification.
  • a suitable cell line for the methods for producing proteinaceous molecules according to the invention is PER.C6, deposited under No. 96022940 at the European Collection of Animal Cell Cultures at the Center for Applied Microbiology and Research.
  • the invention moreover provides methods for producing a proteinaceous molecule comprising a predetermined post- translational modification, said method comprising the steps of: providing a mammalian cell obtainable by a method according to the present invention, with a nucleic acid encoding said proteinaceous molecule in such a way that said mammalian cell harbors said nucleic acid in an expressible form; culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule, and purifying said proteinaceous molecule from the mammalian cell culture.
  • the present invention provides methods for producing a proteinaceous molecule comprising a predetermined post-translational modification, said method comprising the steps of: providing a mammalian cell obtainable by a method according to the present invention, with a nucleic acid encoding said proteinaceous molecule in such a way that said mammalian cell harbors said nucleic acid in an expressible form; culturing said mammalian cell under conditions conducive to the production of said proteinaceous molecule; analyzing said post-translational modifications on said proteinaceous molecule so produced; and determining whether said post-translational modification present on said proteinaceous molecule comprises said predetermined post-translational modification .
  • said methods for producing proteinaceous molecules comprise the extra step of purifying said proteinaceous molecule from the mammalian cell culture. More preferred are methods for producing a proteinaceous molecule in a mammalian of the invention, wherein said mammalian cell is immortalized, and wherein said immortalization is brought about as discussed above. Immortalization can take place prior to the identification of the obtained mammalian cell, but might also take place after the proper cell for proper post-translational modifications is identified, selected and/or obtained.
  • Purification as used herein might be performed by using conventional methods that have been described in the art, however, it is preferred to use purification methods that comprise a step in which the post-translational modifications present in and/or on said proteinaceous molecules are employed. Even more preferred are purification methods that comprise a step in which the predetermined post-translational modifications present in and/or on said proteinaceous molecules are employed.
  • affinity purification methods it is preferred to use antibodies or other binders, such as lectins specific for particular carbohydrate moieties and that are directed against certain types of post-translational modifications. Examples of such antibodies are antibodies directed against (sialyl) Lewis x structures, lacdiNac structures or GalNac Lewis x structures.
  • Using such antibodies enables one to purify the (recombinant) proteins such that a high percentage of the purified protein carries the desired predetermined post-translational modification. Even more preferred are methods in which the proteinaceous molecule is purified to homogeneity. Examples of methods for purification of proteins from mammalian cell culture are provided by the present invention and encompass for instance affinity chromatography methods for the purification of brain-type glycosylated EPO by using antibodies directed against Lewis x structures present in the N-glycans of the recombinantly produced product.
  • the present invention provides a pharmaceutically acceptable composition
  • a pharmaceutically acceptable composition comprising a proteinaceous molecule having a predetermined post-translational modification, obtainable according to methods of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.
  • said proteinaceous molecule in said pharmaceutically acceptable composition is erythropoietin. Even more preferred, said erythropoietin has a lower erythropoietic effect as compared to erythropoietin not having said predetermined post-translational modification.
  • erythropoietin produced in cells with neural protein markers acquires a post- translational modification that is active in neural tissue or on neural cells.
  • the post-translational modifications are not comparable to the post-translational modifications seen on EPO that circulates in the blood.
  • the erythropoietic effects of the EPO produced on cells with the neural protein markers is significantly lower, most likely due to the absence of a high percentage of sialic acids, and/or to the presence of brain-type features such as Lewis x structures and terminal galactosides .
  • the invention provides recombinant erythropoietin comprising at least one post-translational modification selected from the group consisting of: a sialyl Lewis x structure, a Lewis x structure, a l,3- linked fucose attached to N-acetyl-glucosamine, a LacdiNAc structure, a terminal N-acetyl-glucosamine group and a terminal galactose group.
  • said recombinant erythropoietin is produced on a mammalian cell obtainable according to the present invention.
  • Said recombinant erythropoietin could be produced on PER.C6.
  • the present invention further provides the use of PER.C6 for the production of a proteinaceous molecule comprising a predetermined post-translational modification, wherein it is preferred that said proteinaceous molecule is rapidly cleared from the blood and/or used in high dosage.
  • high dosage may be used to treat or prevent acute damage associated with hypoxia, while limiting the adverse side effects of erythropoiesis.
  • the proteinaceous molecules of the present invention are suitable for the treatment of a human or a human body by surgery, therapy or diagnosis.
  • said proteinaceous molecules such as EPO are used for the manufacture of a medicament for the treatment of hypoxia- induced disorders, neurodegenerative afflictions, or acute damage to the central- or peripheral nervous system.
  • said proteinaceous molecules such as EPO are used for the manufacture of a medicament for the treatment of ischemia/reperfusion injuries.
  • said proteinaceous molecules such as EPO are used for the manufacture of a medicament for the treatment of immune disorder and/or inflammatory disease.
  • the invention is particularly useful for the production of proteins that require co-translational and/or post- translational modifications such as glycosylation and proper folding and relates furthermore to the use of human cells capable of producing brain-type co- and/or post- translational modifications on proteinaceous molecules. These cells can for instance be used for the production of human glycoproteins with neural features that might be therapeutically beneficial, due to their neural features.
  • the present invention describes the use of a human cell line with neural characteristics that modifies recombinantly expressed proteins with neural properties such as 'brain-type' or 'neural-type' post-translational modifications such as glycosylation, phosphorylation or folding.
  • PER.C6TM U.S. Pat. No. 6,033,908
  • PER.C6 cells have proven to be particularly suitable for the production of recombinant human proteins, since high yields of proteins such as the human EPO and fully human monoclonal antibodies could be obtained (described in WO 00/63403) .
  • the advantage of using PER.C6 for the production of recombinant proteins is the high level of production and the fact that the cells can be cultured either in an adherent fashion or in suspension in medium without serum or serum-derived components to very high densities .
  • PER.C6 cells express neural marker proteins, by showing that PER.C6 cells can be stained with specific antibodies against vimentin, synaptophysin, neurofilament, glial fibrillary acidic protein (GFAP) , and neural cell adhesion molecules such as N-CAM and CD56.
  • GFAP glial fibrillary acidic protein
  • N-CAM glial fibrillary acidic protein
  • the presence of these marker proteins, as well as the morphological characteristics of the cells indicate that PER.C6 cells are of neural origin.
  • the invention further discloses that recombinant proteins produced by PER.C6 cells can acquire certain tissue specific features such as neural characteristics (e.g., post-translational modifications such as glycosylation) .
  • tissue specific features such as neural characteristics (e.g., post-translational modifications such as glycosylation) .
  • This is exemplified by the production of a protein that harbors so-called brain-type oligosaccharides.
  • human EPO produced by PER.C6 cells is modified with N-linked sugars that significantly differ from the N-linked sugars found in human urinary EPO or in recombinant human EPO produced by Chinese Hamster Ovary (CHO) cells or Baby Hamster Kidney (BHK) cells.
  • Human urinary EPO and recombinant human EPO produced in CHO and BHK cells contain glycosylation structures that can be referred to as 'renal-type' or 'serum-type' oligo- saccharides.
  • the N-linked sugars of these CHO- and BHK-EPO preparations are highly branched, highly galactosylated, and highly sialylated, whereas they lack peripheral ⁇ l,3-linked fucose (Tsuda et al. 1988; Takeuchi et al. 1988; Nimtz et al. 1993; Watson et al. 1994; Rahbek- Nielsen et al . 1997).
  • the nature of the oligosaccharides linked to human EPO produced on PER.C6 has been elucidated and shows that the oligosaccharides of PER. C6-produced human EPO differ significantly from the oligosaccharides present in human urinary EPO and recombinant human EPO produced in CHO and BHK cells.
  • the average sialic acid content of the oligosaccharides of PER.C6-produced human EPO is significantly lower than the average sialic acid content of human urinary EPO or recombinant human EPO (from CHO and BHK) .
  • Brain-type EPO does not circulate in the blood and it might very well be the reason that this EPO form does not need heavy sialylation for its protection against clearance.
  • the very low sialic acid content in PER.C6- produced human EPO is indicative of the presence of N- linked oligosaccharides that contain terminating galactose and/or N-acetyl-galactosamine and/or N-acetyl-glucosamine.
  • N-acetyl-galactosamine is found in significant amounts in the N-linked sugars of PER.
  • N-acetyl-galactosamine is almost absent in the N-linked sugars of human urinary EPO and recombinant human EPO produced by CHO cells. Only trace amounts of N-acetyl- galactosamine have been reported to occur in the N-linked sugars in a few batches of recombinant human EPO produced in BHK cells (Ni tz et al . 1993). Third, the N-linked sugars of human EPO produced in PER.C6 cells are found to contain a very high amount of fucose.
  • Lewis x structures have never been reported to occur in human urinary EPO or in recombinant human EPO produced in CHO and BHK cells.
  • the (sialyl) Lewis x structures present on EPO might as a consequence relate to a role in EPO binding to selectins and a further role in cardioprotection. Such structures might be beneficial and perhaps indicative for a possible role for EPO in direct cardiac effects as discuused above.
  • C ⁇ -produced human EPO have the strong characteristics of brain-type oligosaccharides .
  • PER.C6-produced human EPO has physicochemical properties that differ significantly from human urinary EPO and recombinant human EPO produced by CHO and BHK cells (Toyoda et al. 2000) .
  • PER.C6- produced human EPO is less charged than human urinary EPO and recombinant human EPO produced by CHO and BHK cells due to a lower sialic acid content and it is more hydrophobic due to the very high fucose content.
  • the average pi of PER is less charged than human urinary EPO and recombinant human EPO produced by CHO and BHK cells due to a lower sialic acid content and it is more hydrophobic due to the very high fucose content.
  • C6-produced human EPO is significantly higher than the average pi of human urinary EPO or recombinant human EPO produced by CHO and BHK cells. Because the glycans of EPO, in particular the sialic acids, also have an influence on the binding to the EPO receptor, it is expected that PER. C6-produced human EPO has a different affinity for the EPO receptor than human urinary EPO and recombinant human EPO produced by CHO and BHK cells.
  • the present invention furthermore discloses the use of brain-type proteins produced in neural human cells for the treatment of ischemia/reperfusion injury in mammals and especially in humans.
  • Ischemia/reperfusion injury as used herein is defined as the cellular damage that occurs after reperfusion of previously viable ischemic tissues. Ischemia/reperfusion injury is associated with, for example, but not limited to thrombolytic therapy, coronary angioplasty, aortic cross clamping, cardiopulmonary bypass, organ or tissue transplantation, trauma and shock.
  • the present invention provides the use of therapeutic proteins, produced in mammalian cells, with brain-type oligosaccharides.
  • These brain-type oligosaccharides comprise in particular Lewis x structures, sialyl Lewis x structures, or derivatives thereof containing the (sialyl) Lewis x structure, for the treatment of ischemia/reperfusion injury in mammalian subjects such as humans.
  • the presence of (sialyl) Lewis x structures on recombinant proteins targets these proteins to the injured site of ischemia/reperfusion and thereby exerting their ischemia/reperfusion protective effect more effectively than proteins containing no (sialyl) Lewis x structures.
  • EPO Erythropoietin
  • An advantage provided by the present invention is that PER.C6-produced human EPO is less active in stimulating erythropoiesis in vivo than serum-type human EPO such as recombinant human EPO produced in CHO and BHK cells, which is currently used as a therapeutic drug to treat anemia in human beings.
  • serum-type human EPO such as recombinant human EPO produced in CHO and BHK cells
  • PER. C6-produced human EPO causes a smaller increase in red blood cell production than the highly sialylated fraction of recombinant human EPO that is produced in CHO or BHK cells.
  • PER.C6- produced human EPO causes a smaller increase in the hematocrit value in vivo than the highly sialylated fraction of recombinant human EPO produced in CHO and BHK cells, when applied intravenously at the same dose.
  • the poor effect of PER.C ⁇ -produced human EPO on erythropoiesis is most likely due to a relatively short half-life of the protein in the blood circulation, and/or due to an impaired targeting signal that directs the protein to erythroid progenitor cells in the bone marrow, and/or due to a low affinity of the protein for the EPO-R on erythroid progenitor cells.
  • the impaired functioning of PER.C6- produced human EPO to stimulate erythropoiesis is a direct effect of its oligosaccharide composition, which is, as described above, significantly different from the oligosaccharide composition of serum-type EPO such as human urinary EPO and recombinant human EPO produced in CHO and BHK cells.
  • PER.C ⁇ -produced human EPO has a neurotrophic activity.
  • PER.C ⁇ -produced EPO gives the EPO protein physicochemical and/or pharmacokinetic and/or pharmacodynamic advantages in functioning as a neurotrophic and/or neuro-protecting agent.
  • PER. C6-produced EPO has higher affinity for neural cells and for the EPO-R on neural cells than the highly sialylated serum-type glycosylated human recombinant EPO produced in CHO and BHK cells.
  • Recombinant human EPO produced on non-neural cells (Goto et al. 1988) has a lower affinity for the EPO-R on neural cells than for the EPO-R on erythroid progenitor cells (Musada et al. 1993 and 1994) .
  • EPO neuroprotective role
  • recombinant human EPO as neuroprotective therapy in response to toxic chemicals that may be induced by inflammation or by hypoxia and/or ischemia, or in neurodegenerative disorders.
  • a major drawback is that when applied as a neuroprotective agent, recombinant EPO present in the blood circulation will also give rise to an increase of the red blood cells mass or hematocrit. This, in turn, leads to a higher blood viscosity, which may have detrimental effects in brain ischemia (Wiessner et al. 2001).
  • the present invention provides a solution for the problem that recombinant human EPO that has been applied thus far as a neuroprotective agent has the undesired haematotropic side effect (Wiessner et al . 2001).
  • PER. C6-produced brain-type glycosylated recombinant human EPO has a high potential as a neurogenesis and/or a neuroprotective agent whereas it has a low potential in stimulating erythropoiesis.
  • PER.C ⁇ -produced EPO can be administered systemically (intra-venous, intra-peritoneal, intra-dermal) to inhibit, to prevent and/or to repair the neural damage that is caused by, for example, acute head and brain injury or neuro-degenerative disorders.
  • the present invention also provides products that can be used to modulate the function of tissues that might get heavily damaged by hypoxia, such as the central- and peripheral nervous system, retinal tissue and heart tissue in mammals. Such tissues may be diseased but may also be normal and healthy.
  • Disorders that can be treated by products provided by the present invention may result from acute head-, brain- and/or heart injuries, neuro-degenerative diseases, seizure disorders, neurotoxin poisoning, hypotension, cardiac arrest, radiation, multiple sclerosis and/or from injuries due to hypoxia.
  • Hypoxia may be the result of prenatal- or postnatal oxygen deprivation, suffocation, emphysema, septic shock, cardiac arrest, choking, near drowning, sickle cell crisis, adult respiratory distress syndrome, dysrythmia, nitrogen narcosis, post-surgical cognitive dysfunction, carbon monoxide poisoning, smoke inhalation, chronic obstructive pulmonary disease anaphylactic shock or insulin shock.
  • Seizure injuries include, but are not limited to, epilepsy, chronic seizure disorder or convulsions.
  • the pathology is a result from neuro-degenerative diseases the disorder may be due to AIDS dementia, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, stroke, cerebral palsy, spinal cord trauma, brain trauma, age-related loss of cognitive function, amyotrophic lateral sclerosis, alcoholism, retinal ischemia, glaucoma, general neural loss, memory loss or aging.
  • Other examples of diseases that may be treated with products provided by the present invention include autism, depression, anxiety disorders, mood disorders, attention deficit hyperactivity disorder (ADHD) and cognitive dysfunction.
  • ADHD attention deficit hyperactivity disorder
  • PER.C6-EPO can passively cross the blood-brain barrier in case of blood-brain barrier dysfunction. In case the blood-brain barrier is intact, PER.C6-EPO might be actively transported over the blood-brain barrier through the EPO-R. Some studies suggested that EPO in itself is able to cross the blood-brain barrier when high doses of recombinant EPO is administered (WO 00/61164) . Another possible route for recombinant PER.C6-EPO to cross the blood-brain barrier is via the interaction of the Lewis x glycan structures present on the PER.C6-produced EPO with E-selectin molecules present on human brain microvessel endothelial cells (Lou et al. 1996).
  • E-selectin and EPO may facilitate the transport of EPO across the cerebral endothelial barrier since E-selectin also has been implicated in the migration of T lymphocytes into the CNS (Wong et al . 1999).
  • PER.C6-produced EPO can be administered at a significantly higher dose than serum-type EPO, because PER.C6-EPO will induce erythropoiesis much less efficiently, such that the detrimental effects of the increase in hematocrit is reduced or even absent.
  • PER.C6-EPO can be administered intrathecally by infusion, or through an indwelling ventricular catheter, or through lumbar injection, to inhibit or prevent neural damage.
  • brain-type EPO over serum-type EPO is that in the event of leakage into the blood circulation in the case of blood-brain barrier dysfunction, due to for instance stroke, no undesired side-effects with respect to erythropoiesis will occur.
  • the present invention establishes that indefinitely growing transformed cells that grow to very high densities under serum-free conditions and that have strong neural characteristics, such as PER.C6, are extremely useful to produce factors that depend for their functionality on these characteristics.
  • erythropoietin-like molecules having on average a lower sialic acid residue count per protein backbone are still effective in the treatment and/or prevention of various disorders.
  • EPO with a low sialic acid content is about as potent in reducing infarct size in an experimentally induced stroke in rats as EPO with a higher sialic acid content. It is well established in the art that a high sialic acid content of EPO correlates to longer circulatory half-lifes and increased erythropoietic potential in vivo (Tsuda et al . 1990; Morimoto et al . 1996).
  • the invention provides the use of a composition of erythropoietin-like molecules selected from the group consisting of one or more muteins of erythropoietin, one or more derivatives of erythropoietin, and a composition of one or more fractions of erythropoietin molecules sialylated to a varying degree, for the preparation of a medicament for the treatment of a disorder selected from the group consisting of ischemia, a reperfusion injury, a hypoxia-induced disorder, an inflammatory disease, a neurodegenerative disorder, and acute damage to the central- or peripheral nervous system, wherein said composition of erythropoietin-like molecules has on a protein content basis a lower erythropoietic activity in vivo than epoetin alfa.
  • Embodiments of the invention comprise compositions and use thereof wherein said erythropoietic activity in vivo is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% lower than that of epoetin alfa (Eprex) .
  • Erythropoietin-like molecules are meant to include molecules that have a protein backbone that is identical to or similar to the presently known forms of EPO, e.g. EPO muteins, EPO derivatives, or EPO molecules differing in glycosylation of the protein backbone in qualitative and/or quantitative respect.
  • Muteins as used herein are meant to consist of erythropoietin-like molecules that have one or more mutations in the protein backbone by deletion, addition, substitution and/or translocation of amino acids relative to the protein backbone of epoietin alfa and shall include naturally occurring allelic variants as well as genetically and/or chemically and/or enzymatically obtained variants. Such molecules should still be able to confer a functional activity of EPO. They are obtainable using standard techniques of molecular biology, well known to those of skill in the art.
  • a derivative as used herein is an erythropoietin-like molecule that is obtainable from erythropoietin or epoietin alfa, or any other functional mutein of epoietin alfa by the chemical or enzymatic modification thereof.
  • Erythropoietic activity as meant herein is the stimulatory effect of EPO on red blood cell production in a human or animal subject, as can be measured by the increase in hematocrit values at a certain point in time after administration to the human or animal subject of erythropoietin-like molecules (e.g see example 9), or the measuring the hemoglobin concentration.
  • Epoetin alfa is the recombinant human EPO form present in currently marketed Eprex-TM, and is similar or identical (with respect to amino acid and carbohydrate composition) to human erythropoietin isolated from urine of anemic patients . Treatment regimes for erythropoietic purposes are well established. In general EPO dosages are given in IU (international units), referring to the activity of EPO in erythropoiesis. Such IU correlate to the protein content of EPO but are operationally defined, and hence the correlation may vary between different batches . As a rule of thumb, one IU corresponds to 8-10 ng epoetin alfa.
  • the erythropoietic activity of the erythropoietin-like molecules is referred to on a protein content basis, to get rid of the variable introduced by defining IU.
  • the concentration of EPO molecules in such preparations can easily be defined according to standard procedures. This will allow to determine the relative specific activity e.g in IU/g (see e.g. EP 0428267).
  • Storring et al. (1992) examples of other forms of EPO currently on the market are Procrit or Epogen (both epoetin alfa) and
  • Aranesp (darbepoetin alfa, EPO with extra N-glycosylation sites to increase circulatory half-life and erythropoietic activity) .
  • the erythropoietic activity may vary somewhat between the various commercial epoetin alfa preparations on the market, they are generally optimized for high erythropoietic activity.
  • the present invention discloses the use of EPO-like molecules or EPO-forms that have a lower hemopoietic or erythropoietic activity, thereby diminishing or avoiding the side-effects of increased erythropoiesis when this is not desired.
  • a composition of erythropoietin-like molecules is characterized by an average number of sialic acid residues per erytropoetin-like molecule that is at least 10% lower than the average number of sialic acid residues per erythropoietin molecule in epoetin alfa.
  • said average number of sialic acid residues may be chosen to be at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% lower than the average number of sialic acid residues per EPO protein backbone in epoetin alfa.
  • Said average number of sialic acid residues in the erythropoietin-like molecule preferably lies between between 0 and 90% of the average number of sialic acid residues per EPO molecule in epoetin alfa, but the exact percentage may depend from disorder to disorder, and - sometimes - from patient to patient, as some patient - disorder combinations are less vulnerable to high hematocrit values than others.
  • the number of sialic acid residues could be described per EPO-like molecule, e.g. 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 sialic acid residues per EPO-like molecule.
  • the values are averages calculated for a composition that consists of epo-like molecules of varying degree of sialylation, non-integer values in between the mentioned values are possible to define the molecules according to the invention.
  • the optimal range could be determined empirically.
  • the average number of sialic acid residues per molecule or the sialic acid content of EPO can be determined according to published procedures, and are well known to persons skilled in the art. One possible procedure is described in EP 0428267. In brief, the sialic acid residues are cleaved from the EPO-like molecules by hydrolysis with 0.35 M sulfuric acid at 80°C for 30 minutes, and the solutions are neutralized with sodium hydroxide prior to analysis.
  • the sialic acids can be removed by enzymatic cleavage according to standard procedures.
  • the amount of EPO is estimated using well known procedures e.g. by using commercially available protein assay kits (e.g. Bradford assay, Biorad) and standard curves using recombinant human EPO as a standard, absorbance at 280 nm, ELISA, RIA, and the like.
  • Sialic acid content can be analyzed by the procedure of Jourdian et al. (1971).
  • sialic acids can be analysed using High Performance Anion-Exchange Chromatography, using procedures well known to the skilled person (e.g.
  • the sialic acid content can be expressed as moles of sialic acid per mole of EPO, or an average number of sialic acid residues per EPO-like molecule.
  • An indication for the average number of sialic acid residues per EPO-like molecule can also be given by iso-electric focusing (see example 4), which measures the pi.
  • EPO-like molecules e.g.produced recombinantly in any suitable host cell line
  • enzymes that cleave off the sialic acid in particular such as neuraminidases
  • enzymes that cleave off more substituents (including sialic acid) of the glycosylation structures such as e.g.
  • N-glycanase F (removes whole N-glycan) , endoglycosidase F 2 (removes bi- antennary structures) , endoglycosidase F 3 (removes bi- and tri-antennary structures, and the like, or treatment of EPO-like molecules with chemicals, including but not limited to acids, that results in decrease of the average number of sialic acid residues per EPO-like molecule.
  • a highly sialylated EPO fraction could be thus desialylated and used in the present invention.
  • EPO-like molecules with an average lower number of sialic acid molecules are obtained by purifying or separating such forms from a mixture containing both higher and lower sialylated EPO.
  • the currently used production systems generally result in such mixtures, and EPO that is intended for erythropoietic purposes is prepared by purifying the forms with a high average number of sialic acid residues.
  • the present invention discloses use of other fractions from this process, i.e. the EPO forms with a lower number of sialic acid residues. Purifying or separating such fractions can be done using well-established techniques known to the skilled person, such as ion-exchange, affinity purification, and the like.
  • the erythropoietin-like molecules of the invention are preferably produced recombinantly .
  • This can be done in any suitable expression system, including but not limited to Chinese Hamster Ovary cells, Baby Hamster Kidney cells, human cells, such as HeLa, HEK293 or PER.C6. Expression in lower eukaryotic cells such as insect cells or yeast is also possible.
  • Production of EPO-like molecules having low sialic acid content may be performed on sialylation- deficient cell systems, by way of a natural lack of sialylating enzymes, such as certain prokaryotic hosts, or by mutagenesis or genetic modification of hosts otherwise capable of producing sialylated proteins.
  • the EPO-like molecules are produced by methods according to the invention, thereby producing molecules with a predetermined post-translational modification.
  • the composition comprising erythropoietin-like molecules is characterized by the presence of erythropoietin-like molecules that once administered parenterally to a human or an animal subject are cleared from the bloodstream at a faster rate than epoetin alfa. Clearance from the bloodstream can be measured by methods well known in the art, e.g. by determining the half-life of a protein in blood such as done in example 18. In healthy volunteers epoetin alfa has a circulatory half-life of about 4 hours after repeated intravenous injections. A half-life of about 5 hours in patients with chronic renal insufficiency, and about 6 hours in children has been reported.
  • Erythropoietin-like molecules with a lower ratio of tetra- antennary structures to bi-antennary structures will also have a shorter half life in plasma (Misaizu et al, 1995; Takeuchi et al, 1989) . Production of EPO in cell lines that give rise to such lower ratios is feasible, or alternatively these forms are purified away from the forms containing more tetra-antennary structures. Such compositions comprising relatively more bi-antennary structures are also useful according to the invention.
  • one advantage of the current invention is that higher maximal concentrations of erythropoietin-like molecules in the circulation can be reached as compared to the currently used EPO forms such as Eprex, Procrit, NESP. If high concentrations of EPO-like molecules would be desired for said treatment, this can be done by administering high doses of the compositions of the invention. Administering of similar doses on a protein content basis of the currently used EPO-like molecules would lead to higher erythropoiesis, which is an undesired side-effect for said treatments.
  • the invention also provides pharmaceutical compositions comprising said erythropoietin-like molecules, and methods for treatment or preventing disorders selected from said groups, as wel as compositions of erythropoietin-like molecules for for the preventative and/or therapeutic treatment of the human or animal body.
  • Example 1 Studies on expression of marker proteins in PER.C6TM cells.
  • Retinas generally comprise a number of different cells types (at least 55 different neural subtypes) , including neural and fibroblast-like cells (Masland 2001) .
  • a study was performed to test the expression of marker proteins in or on the cells. These markers are known in the art to be characteristic for certain cell types and/or tissues. The marker proteins are given in Table I.
  • Marker protein expression was tested using antibodies directed against the marker proteins. In each experiment, a negative control (PER.C6 cells not incubated with antibody) and a positive control were taken along. These positive controls are sections of human tissue known to express the marker protein (Table II) .
  • PER.C6 cells were cultured on glass slides in a medium chamber (Life Technologies, Nunc Lab-Tek, Chamber Slide, radiation sterilized, 2 medium chambers, cat.no. 154464A) .
  • PER.C6 cells were seeded at 65-70% confluency (2 wells per culturing chamber) and cultured for 24 h at 37°C (10% C0 2 , 95% air) .
  • the medium was aspirated and the glass slides with cells were washed with sterile PBS, removed from the medium chamber and air-dried. Cells were fixed on the glass slides by incubation in acetone for 2 min. After air drying, slides were wrapped in aluminum foil and frozen at a temperature lower than -18 °C until use.
  • Positive control tissues were obtained from banks of tissue slides prepared for routine use at the division of pathology, Academic Hospital Erasmus University (Rotterdam, The Netherlands) . Frozen sections were prepared (5 ⁇ m) and fixed in acetone, according to routine procedures.
  • the primary antibodies, their respective marker proteins, the suppliers and the catalog numbers of the antibodies are given in Table III.
  • the dilutions, also detailed in Table III, are made in Phosphate Buffered Saline (PBS) , 1% Bovine Serum Albumin. Incubations of the slides with the primary antibody were done for 30 min at room temperature, rinsed with PBS and incubated with the secondary antibody.
  • These secondary antibodies were either goat anti rabbit (DAKO E0432; 1:50 dilution) or goat anti mouse (DAKO E0433; 1:50 dilution), depending on the nature of the primary antibody used.
  • the second antibody was conjugated with biotin.
  • C6 cells are of neural origin since the cells stained positive for vimentin, synaptophysin, neurofilament, GFAP and N-CAM.
  • Example 2 Monosaccharide composition of PER. C6-EPO derived N-glycans compared to that of Eprex.
  • a first step in characterizing the N-glycan structures produced by PER.C6 is the measurement of the molar ratio of the various monosaccharides .
  • the monosaccharide analysis was performed using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC- PAD) .
  • Eprex Jansen Cilag
  • PER.C6-EPO samples were purified by affinity chromatography using a column packed with C4 sepharose beads (bedvolume of 4 ml, Amersham Pharmacia Biotech) coupled with mouse monoclonal anti-EPO (IgGl) antibodies.
  • Bound EPO molecules were eluted with 0.1 M glycine-HCl, pH 2.7, and resulting fractions were immediately neutralized by adding sodium/potassium phosphate buffer pH 8.0. Subsequently, the fractions containing EPO were pooled and the buffer was exchanged to 20 mM Tris-HCl, containing 0.1% (v/v) Tween 20, by utilizing Hiprep 26/10 desalting columns (Amersham Pharmacia Biotech) .
  • EPO samples were dialyzed overnight against MilliQ-grade water, and dried in a Speedvac evaporator. Dried EPO samples (quantities ranged from 39 to 105 ⁇ g) were dissolved in incubation buffer (1:1 diluted C3 profiling buffer, Glyko) . Upon addition of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol to final concentrations of 0.1% (w/v) and 0.3% (v/v), respectively, samples were denatured for 5 min at 100 °C.
  • SDS sodium dodecyl sulfate
  • beta-mercaptoethanol Upon addition of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol to final concentrations of 0.1% (w/v) and 0.3% (v/v), respectively, samples were denatured for 5 min at 100 °C.
  • Nonidet P-40 BDH was thereafter added to a final concentration of 0.75% (v/v), and EPO was deglycosylated overnight at 37°C, using N-glycanase F ( U, Glyko) .
  • N-glycanase F U, Glyko
  • released N-glycans were separated from proteins, salts, and detergents by using graphitized carbon black (Carbograph) SPE columns (Alltech) , according to Packer et al. (1998) .
  • the column was run isocratically in 16 mM NaOH (Baker) at a flow rate of 0.25 ml/min.
  • the monosaccharide composition was calculated by comparing the profile with that obtained with a mixture of monosaccharide standards that consisted of fucose, deoxyglucose, galactosamine, glucosamine, galactose, and mannose.
  • the monosaccharide analysis clearly showed that the glycosylation status of PER.C6-EPO is significantly different from Eprex (Table V) .
  • GalNAc ⁇ l-4GlcNAc GalNAc ⁇ l-4GlcNAc
  • Another striking feature of PER.C6-EPO is the relative abundance of fucose residues shown in Table V. This strongly indicates the presence of Lewis structures in the N-glycans of PER.C6-EPO. In contrast, Eprex is known to be devoid of Lewis structures. Consequently, the amount of fucose found in Eprex can be solely attributed to N-glycan core fucosylation. Notably, the data from the monosaccharide analyses also demonstrated that culture conditions affect the glycosylation status of EPO in PER.C6. It should not be concluded that the culture conditions are solely responsible for the predetermined post-translational modifications that are present on the proteins produced.
  • the cell lines should be able to modify the post-translational modifications of the proteins produced on such cells through the presence of certain specific glycosylation enzymes such as transferases .
  • the culture conditions can only exert additive activities. For instance, when the EPO-producing clones were cultured (in suspension) in JRH Excell 525 medium, the N-linked glycans of EPO were found to contain higher levels of GlcNAc, GalNAc, Gal, and Fuc as compared to the N-linked sugars of EPO derived from cultured (adherent) cells in DMEM (Table V) . This effect was particularly evident in the case of clone P8.
  • the elevated level of GlcNAc may suggest that the branching of the N- linked sugars is increased and/or that the N-linked sugars contain more lactosamine repeats, when cells are cultured in JRH medium.
  • the increase in N-acetyl glucosaminylation and in (N-acetyl-) galactosylation in turn gives rise to an increased number of fucose-acceptor sites thereby providing an explanation for the increase of the Fuc content.
  • Example 3 Mass spectro etric analysis to reveal structural differences between N-glycans of PER.C6-EPO and Eprex.
  • the N-linked glycans were released from the EPO pools by N-glycanase F treatment and desialylated by neuraminidase treatment.
  • Eprex was analyzed in parallel as a reference. Representative mass spectra of the various EPO samples are shown in Fig. 1A-G: Eprex and the purified, fractionated (pools A, B, and C from the anion exchange chromatography column).
  • PER.C6-EPO samples derived from the indicated clones cultured in DMEM were treated with glycanase F and neuraminidase, and thereafter analyzed by MALDI-MS.
  • Oligosaccharides with (sialylated) Lewis x epitopes are known as essential recognition sequences for selectins, mediating cell-cell adhesions in both inflammatory and immune responses (Varki et al. 1999) and are characteristically found in brain glycoproteins (Margolis and Margolis 1989) .
  • numerous glycoproteins carrying these Lewis x structures have been shown to have therapeutic potential by exhibiting anti-inflammatory and immunosuppressive activities. It is emphasized here that a mass signal cannot always be unambiguously assigned to a certain sugar structure, as e.g. residues, like GlcNAc and GalNAc, have the same mass.
  • peaks represent N-glycans with so-called LacdiNAc (e.g., GalNAc ⁇ l-4GlcNAc) structures.
  • LacdiNAc e.g., GalNAc ⁇ l-4GlcNAc
  • peaks with m/z values of ⁇ 2038 and ⁇ 2185 most likely represent N-glycans with LacdiNAc motifs. Otherwise, these peaks would represent tetra-antennary structures, which terminate in GlcNAc due to the absence of Gal or GalNAc.
  • the presence of the proximal Fuc implies that the sugar contained a Gal or GalNAc residue that is necessary to form a motif that is recognized by the fucosyltransferase (FUT) that catalyzes the formation of the Lewis structure.
  • FUT fucosyltransferase
  • the relative occurrence of the different sugars varies between the EPO preparations derived from two independent PER.C6 clones as judged by the difference in the relative height of certain peaks.
  • the putative bi- antennary sugars with LacdiNAc motifs Fig.
  • Example 4 Comparison of sialic acid content of PER.C6-EPO and CHO-EPO.
  • the sialic acid content of PER.C6-EPO was analyzed and compared with erythropoietin derived from Chinese Hamster Ovary cells (CHO-EPO) by iso-electric focusing (IEF) using IPG strips (Amersham Pharmacia Biotech) that have a linear pH gradient of 3-10. After the focusing, the EPO isoforms were passively blotted onto nitrocellulose, and visualized using an EPO-specific antibody and ECL (Fig. 2) .
  • EPO made by four different PER.C6 clones (lanes C, D, E, and F) , and three different CHO clones stably expressing EPO (lanes G, H, and I) were analyzed by iso-electric focusing to determine the sialic acid content.
  • the EPO producing CHO and PER.C6 cell lines were generated generally according to methods described in WO 00/63403 using the Neomycine- resistance gene as a selection marker.
  • One thousand eU of PER.C6-EPO and 500 eU of CHO-EPO were loaded per strip.
  • Eprex Five hundred IU of Eprex (lane A) and neuraminidase-treated (partially desialylated) Eprex (lane B) were used to identify the various EPO isoforms. After focusing, EPO was blotted onto nitrocellulose filter and visualized using a monoclonal antibody against EPO and ECL.
  • the Eprex sample representing a commercially available EPO is a formulation containing highly sialylated isoforms and was used as a marker. The results clearly demonstrated that CHO cells are able to make EPO isoforms containing up to at least 12 sialic acids per molecule (lanes G-I), confirming data by Morimoto et al. (1996) .
  • Example 5 ⁇ l,3-, ⁇ l,6- and ⁇ l,2-fucosyltransferase activities on PER.C6 cells.
  • the glycosylation potential of a cell is largely determined by an extensive repertoire of glycosyl- transferases involved in the step-wise biosynthesis of N- and O-linked sugars.
  • the activity of these glycosyl- transferases varies between cell lines and, hence, glycoproteins produced in different cell lines acquire different glycans.
  • FUTs fucosyltransferases
  • the activities of the indicated FUTs in cell-extracts of PER.C6 and CHO were measured using a glycosyltransferase activity assay.
  • This assay measures the glycosyltrans- ferase-catalyzed reaction between a saccharide (in this case fucose) and a sugar substrate.
  • the GalT activity was also measured as an internal control.
  • the values represent the mean values from two experiments. All values, and in particular those of PER.C ⁇ were 2-3 fold lower in the second experiment. Notably, the activities were expressed per mg protein (present in the cell extract) . Because PER.C6 cells are significantly bigger than CHO cells, the differences between the FUT and GalT activities of CHO and PER.C ⁇ cells may be bigger or smaller than they appear.
  • Example 6 Glycans with Lewis x epitopes present on PER.C6- EPO. Because PER.C ⁇ possesses ⁇ l,3-, but no ⁇ l,2- fucosyltransferase activity, it is very likely that PER.C ⁇ produced N-glycan chains which contain Lewis x instead of Lewis y epitopes. We verified this by labeling PER.C6-EPO with a mouse monoclonal antibody (Calbiochem) that specifically recognizes Lewis x structures, using western blotting.
  • Calbiochem mouse monoclonal antibody
  • Equal amounts of PER.C6-EPO (derived from clone P7, here indicated as P7.100) and Eprex, untreated (-) or treated with HCl (+), were run on a SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane using methods known to persons skilled in the art.
  • a monoclonal antibody anti-mouse IgM, Calbiochem
  • ECL Anamersham Pharmacia Biotech
  • Example 7 Lewis x structures expression at cell surface of PER.C6 cells.
  • Example 8 Inhibition of apoptosis by PER.C6-EPO (brain- type) in vitro, in NT2 cells and hNT cells cultured under hypoxic conditions .
  • PER.C ⁇ -produced (brain-type) EPO and serum-type EPO are compared in their in vitro activity to protect rat-, mouse- and human cortical neural cells from cell death under hypoxic conditions and with glucose deprivation.
  • neural cell cultures are prepared from rat embryos as described by others (Koretz et al . 1994; Nagayama et al . 1999; White et al. 1996) .
  • the cells are maintained in modular incubator chambers in a water-jacketed incubator for up to 48 h at 37°C, in serum-free medium with 30 mM glucose and humidified 95% air/5% C0 2 (normoxia) or in serum-free medium without glucose and humidified 95% N 2 /5% C0 2 (hypoxia and glucose deprivation) , in the absence or presence of 30 pM purified PER.C6-produced brain-type EPO or 30 pM Eprex.
  • the cell cultures are exposed to hypoxia and glucose deprivation for less than 24 h and thereafter returned to normoxic conditions for the remainder of 24 h.
  • the cytotoxity is analyzed by the fluorescence of Alamar blue, which reports cells viability as a function of metabolic activity.
  • the neural cell cultures are exposed for 24 h to 1 mM L-glutamate or ⁇ -amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) under normoxic conditions, in the absence or presence of various concentrations of purified PER.C ⁇ -produced EPO or Eprex.
  • the cytotoxity is analyzed by the fluorescence of Alamar blue, which reports cell-viability as a function of metabolic activity.
  • the viability of cells treated with PER.C6-EPO is expected to be similar to the viability of cells treated with Eprex.
  • Example 9 Activity of PER.C6-EPO (brain-type) in stimulating erythropoiesis in rats compared to serum-type EPO.
  • the potential of recombinant human EPO to stimulate the production of red blood cells can be monitored in a rodent model that has been described by Barbone et al . (1994). According to this model, the increase in the reticulocyte counts is used as a measure for the biological activity of the recombinant human EPO preparation.
  • Reticulocytes are the precursors of red blood cells and their production, in response to EPO, can be used as a measure for the potential of EPO in stimulating the production of red blood cells.
  • An increased production of red blood cells leads to a higher hematocrit value.
  • PER.C6-EPO The activities of PER.C6-EPO and Eprex were compared in six groups of three Wag/Rij rats.
  • Various doses of PER.C6-EPO (P7-EPO) , Eprex and diluent buffer as a negative control were injected intravenously in the penile vein at day 0, 1, and 2.
  • PER.C6-EPO was administered at a dose of 5, 25, or 125 eU (Elisa units) as determined by the commercially available EPO-specific R&D Elisa Kit, whereas Eprex was administered at a dose of 1 or 5 eU.
  • All EPO preparations were diluted to the proper concentration in PBS/0.05% Tween 80 in a total volume of 500 ⁇ l .
  • 250 ⁇ l of EDTA blood was sampled by tongue puncture.
  • the percentage of reticulocytes in the total red blood cell population was determined.
  • Fig. 6 bars indicate the percentage of reticulocytes present in the total red blood cell population
  • the daily administration of 1 eU of Eprex into the rats, for a total period of three days caused a significant increase in the reticulocyte counts at the fourth day compared to reticulocyte counts in rats that received diluent buffer only.
  • the reticulocyte counts were even more boosted by increasing the Eprex dose five-fold.
  • the reticulocyte counts were clearly less increased using equivalent amounts of PER.C6-EPO.
  • Example 10 Effect of PER.C6-EPO on cerebral ischemia following experiment subarachnoid hemorrhage .
  • PER.C6-EP0 is more effective in neuroprotection during cerebral ischemia than serum-type EPO
  • we compare the effects of systemic adminstration of PER.C ⁇ - produced brain-type EPO and serum-type EPO in a rabbit model of subarachnoid hemorrhage-induced acute cerebral ischemia. Therefore, 32 animals that are divided into 4 groups (n 8) are studied.
  • the experimental subarachnoid hemorrhage is produced by a percutaneous injection of autologous blood into the cisterna magna after anesthesizing the animal. After the injection, the rabbits are positioned in ventral recumbence for 15 min to allow ventral blood-clot formation. Animals of group 2, 3, and 4 are injected with diluent buffer, Eprex, and purified PER.C ⁇ -produced brain-type EPO, respectively, at 5 min after the induction of subarachnoid hemorrhage, and are continued at 8, 16, and 24 h thereafter. All injections are administered intraperitoneally.
  • the diluent buffer consists of serum albumin (2.5 mg/ml), sodium chloride (5.84 mg/ml), anhydrous citric acid (0.057 mg/ml, H 2 0) .
  • the animals are euthanized at 24 h after the subarachnoid hemorrhage, and their brains are removed.
  • the brains are thereafter coronally sectioned at 10-25 ⁇ m in a freezing microtome, starting at the bregma and continuing posteriorly to include the cerebellum (Ireland and MacLeod 1993) .
  • the slices are stained with hematoxylin and eosin.
  • the number of eosinophilic neuronal profiles containing pyknotic nuclei, per high-power microscopic field (lOOx) is determined in five randomly selected sections of the lateral cortex obtained at several coronal levels posterior to the bregma.
  • PER.C6-EPO treated animals are expected to have a lower number of damaged neurons than animals that are not treated or that are treated with a placebo.
  • Example 11 Erythropoietin receptor expression in rat neonatal cardiomyocytes following hypoxia/reoxygenation .
  • RNA is isolated using Trizol (GIBCO) , extracted by chloroform and precipitated by isopropyl alcohol.
  • Trizol Trizol
  • 15 ⁇ g of total RNA is separated on a 1.5% formaldehyde/MOPS-agarose gel, blotted to nitrocellulose, and hybridized with a 32 P-labeled probe for EPO receptor ( ⁇ 400 bp cDNA fragment) .
  • Hybridization takes place overnight at 65°C in phosphate buffer, pH 7.2 and is followed by 2 washes in 2xSSC at room temperature, 2 washes in 0.2xSSC/0.1%SDS at 65°C and 2 washes in 2xSSC at room temperature.
  • Hybridization signals are visualized by exposing the membrane to an X-ray film (Kodak) . Expression levels are corrected for GAPDH mRNA levels.
  • Example 12 The effect of brain-type PER.C6-EPO and serum- type EPO (Eprex) on apoptosis in rat neonatal cardiomyocytes , cultured under hypoxic conditions .
  • the experiment is divided into 4 groups: A) cardiomyocytes cultured under normoxic conditions (95% air/5% C0 2 ) ; B) cardiomyocytes cultured under hypoxia/reoxygenation conditions in the presence of 30 pM purified PER.C6- produced EPO;
  • Apoptosis is quantified by morphological analysis, DNA laddering and by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) .
  • TUNEL terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling
  • myocytes monolayers are fixed and stained with Hoechst 33324.
  • the morphological features of apoptosis are monitored by fluorescence microscopy. At least 400 cells from 12 randomly selected fields per dish are counted.
  • DNA laddering characteristic for apoptosis
  • cardiomyocytes are lysed in lysis buffer and electrophoresed on 2% agarose gel. The gel is stained with ethidium bromide, and DNA fragments are visualized under ultraviolet light.
  • In situ detection of apoptotic cardiomyocytes is performed by using TUNEL with an in situ cell death detection kit (Boehringer Mannheim) .
  • Example 13 The effect of PER.C6-EPO and serum-EPO on the infarct size in a rat model of myocardial ischemia/ reperfusion .
  • infarct size is determined by differential staining with patent blue violet (5%) and triphenyl tetrazolium chloride (TTC) .
  • the coronary ligature is retightened, and an intravenous injection of patent blue violet is given to stain the normally perfused regions of the heart.
  • the heart is then removed and bathed in ice-cold saline before removal of the atria, great vessels and right ventricle.
  • the left ventricle is sliced into thin sections, and the unstained area at risk (AAR) is separated from the normally perfused blue sections, cut into 1-2 mm 3 pieces, and incubated with TTC. With a dissecting microscope, the necrotic areas (AN, pale) are separated from the TTC- positive (brick red-staining) areas. All areas of the myocardium are then weighed individually, and infarct size is calculated.
  • Example 14 Isolation and fractionation of PER.C6-EPO glycoforms employing a high ⁇ l,3-linked fucose content.
  • the fucose-specific Aleuria aurantia lectin (AAL) is used to preferentially purify PER.C6-EPO glycoforms with a high Lewis x and/or sialyl-Lewis x content.
  • AAL Aleuria aurantia lectin
  • This lectin is coupled to CNBr-activated Sepharose 4B beads according to procedures commonly known by a person skilled in the art.
  • PER.C6-EPO that is secreted into the culture medium by human EPO-producing PER.C ⁇ cells is first roughly separated from cell debris and other contaminants by affinity column chromatography using monoclonal antibodies specific for human EPO.
  • the purified EPO is subjected to a second chromatography procedure in which the EPO molecules possessing ⁇ l,3-linked fucose are bound to a column containing the immobilized AAL.
  • EPO glycoforms that lack ⁇ l,3-linked fucose do not bind to the column and are collected in the flow-through.
  • EPO glycoforms carrying ⁇ l,3-linked fucose are eluted from the column by using fucose as a competitor for binding to AAL.
  • EPO glycoforms having a high or low ⁇ l,3-linked fucose content are separately eluted from the column by increasing the fucose concentration step-wise or gradually during the elution.
  • EPO glycoforms with a high ⁇ l,3-linked fucose content are eluted at a higher concentration of fucose than EPO glycoforms with a low ⁇ l,3-linked fucose content.
  • This method enables one to purify erythropoietin from the culture medium by employing the specific characteristics of the post-translational modifications, such as Lewis x structures brought about by the cells in which the protein is produced.
  • Example 15 Isolation and fractionation of PER.C6-EPO glycoforms with a high LacdiNAc content.
  • PER.C6-EPO glycoforms carrying so-called lacdiNAc oligosaccharide structures are specifically isolated by the use of monoclonal antibodies against these lacdiNAc structures.
  • Mouse monoclonal antibodies such as 99-2A5-B,
  • PER.C6-EPO that is secreted into the culture medium by human EPO-producing PER.C ⁇ cells is first roughly separated from cell debris and other contaminants by affinity column chromatography using monoclonal antibodies specific for human EPO.
  • the purified EPO is subjected to a second chromatography procedure in which the EPO molecules carrying lacdiNAc structures are bound to a column containing the immobilized lacdiNAc- specific monoclonal antibodies.
  • EPO glycoforms that lack the lacdiNAc structures do not bind to the column and are collected in the flow-through.
  • EPO glycoforms carrying the lacdiNAc structures are eluted from the column at a low pH or by using GalNAc or synthetic lacdiNAc oligosaccharides as a competitor for binding to the lacdiNAc specific antibodies .
  • EPO glycoforms carrying a relatively high percentage of lacdiNAc structures are separately eluted from the column by increasing the GalNAc or lacdiNAc concentration step-wise or gradually during the elution.
  • EPO glycoforms with a relatively high percentage of lacdiNAc structures are eluted at a higher concentration of GalNAc or lacdiNAc than EPO glycoforms possessing a relatively low percentage of lacdiNac structures.
  • this method enables one to purify erythropoietin from the culture medium by employing the specific characteristics of the post-translational modifications, such as Lewis x and lacdiNac structures brought about by the cells in which the protein is produced.
  • Example 16 Isolation and fractionation of PER. C6-EPO glycoforms with a high GalNAc-Lewis x content.
  • PER.C6-EPO glycoforms carrying so-called GalNAc-Lewis x oligosaccharide structures are specifically isolated by the use of monoclonal antibodies against these GalNAc-Lewis x structures.
  • Mouse monoclonal antibodies such as 114-5B1- A, 176-3A7, 290-2D9-A, and 290-4A8 (Van Remoortere et al . 2000) specifically recognize GalNAc-Lewis x structures and are purified and coupled to CNBr-activated Sepharose 4B beads according to procedures commonly known by persons skilled in the art.
  • PER.C ⁇ -EPO that is secreted into the culture medium by human EPO-producing PER.C ⁇ cells is first roughly separated from cell debris and other contaminants by affinity column chromatography using monoclonal antibodies specific for human EPO. Thereafter, the purified EPO is subjected to a second chromatography procedure in which the EPO molecules carrying GalNAc-Lewis x structures are bound to a column containing the immobilized GalNAc- Lewis x specific monoclonal antibodies. EPO glycoforms that lack the GalNAc-Lewis x structures do not bind to the antibodies attached to the column and are collected in the flow-through.
  • Bound EPO glycoforms carrying the GalNAc- Lewis x structures are eluted from the column at low pH or by using synthetic GalNAc-Lewis x as a competitor for binding to the GalNAc-Lewis x specific antibodies.
  • EPO glycoforms carrying a high GalNAc-Lewis x content can be separately eluted from the column by increasing the concentration of GalNAc-Lewis x competitor step-wise or gradually during the elution.
  • EPO glycoforms with a high GalNAc-Lewis x content are eluted at a higher concentration of GalNAc-Lewis x than EPO glycoforms possessing a low GalNAc-Lewis x content.
  • this method enables one to purify EPO from the culture medium by employing the specific characteristics of the post-translational modifications, such as Lewis x, lacdiNac or GalNac-Lewis x structures brought about by the cells in which the protein is produced. This does however, not imply that other modifications with the (predetermined) post-translational modifications cannot be employed for proper purification of the protein.
  • EPO EPO
  • the present invention is not limited to production and/or purification of EPO with brain-type characteristics.
  • Various other (human) therapeutic and/or diagnostic peptides and proteins which may find use in treating disorders of the brain and other parts of the central- and peripheral nervous system and/or other ischemic/reperfusion damaged tissues, can be produced by means and methods of the present invention.
  • EPO with a low sialic acid content has a similar potency as EPO with a high sialic acid content in reducing the infarct size after middle cerebral artery occlusion in rats .
  • the effect of PER.C6-EPO and Eprex on the size of a brain infarct which was experimentally induced by the occlusion of the middle cerebral artery (MCA) , was studied in Wistar male rats weighing 200-250g, using a method similar to the method published by Siren et al . , 2001.
  • the right carotid artery of the animals was permanently occluded whereas the MCA was reversibly occluded for 60 min using a metal clip.
  • PER.C6-EPO with an average sialic acid content of ⁇ 6 sialic acids per molecule or Eprex ( ansen-Cilag; commercially available EPO) with an average sialic acid content > 9 sialic acids per molecule
  • Eprex ansen-Cilag; commercially available EPO
  • the sialic acid content of the PER.C6-EP0 preparation ranged from 0-9 sialic acids per molecule whereas Eprex contained more than 8 sialic acids per molecule.
  • the occlusion was terminated by the removal of the metal clip surrounding the MCA.
  • Example 18 Determination of half-life of EPO in rats .
  • the decrease in the concentration of Eprex in the plasma displays a bi-phasic curve representing a distribution phase and a clearance phase.
  • Eprex had a half-life of about 180 min during the clearance phase.
  • the half-life of PER.C ⁇ -EPO is measured using the same protocol .
  • Butcher EC (1991) Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033-1036
  • Erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.
  • Silverberg DS Wexler D, Blum M, Keren G, Sheps D,

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Abstract

La présente invention concerne des protéines et leurs méthodes de production. Les protéines de l'invention contiennent des modifications post-traductionnelles spécifiques. L'invention concerne en particulier des érythropoïétines contenant une faible quantité de résidus d'acide sialique autres que l'époétine alfa.
PCT/NL2002/000257 1999-04-15 2002-04-19 Methodes et moyens de production de proteines avec des modifications post-traductionnelles preetablies Ceased WO2003089468A1 (fr)

Priority Applications (30)

Application Number Priority Date Filing Date Title
AU2002307635A AU2002307635A1 (en) 2002-04-19 2002-04-19 Methods and means for producing proteins with predetermined post-translational modifications
PCT/NL2002/000257 WO2003089468A1 (fr) 2002-04-19 2002-04-19 Methodes et moyens de production de proteines avec des modifications post-traductionnelles preetablies
MXPA04003940A MXPA04003940A (es) 2001-10-29 2002-10-29 Metodos y medios para producir proteinas con modificaciones postraduccionales predeterminadas.
CA2465007A CA2465007C (fr) 2001-10-29 2002-10-29 Procedes et moyens de fabrication de proteines presentant des modifications post-traductionnelles predeterminees
AT02770322T ATE542904T1 (de) 2001-10-29 2002-10-29 Verfahren und mittel zur herstellung von proteinen mit vorbestimmten posttranslationalen modifikationen
PCT/NL2002/000686 WO2003038100A1 (fr) 2001-10-29 2002-10-29 Procedes et moyens de fabrication de proteines presentant des modifications post-traductionnelles predeterminees
KR1020067008079A KR100602772B1 (ko) 2001-10-29 2002-10-29 예비결정된 번역후 변형을 갖는 단백질을 생성하기 위한장치 및 방법
EA200400605A EA008220B1 (ru) 2001-10-29 2002-10-29 Композиция, включающая эритропоэтинподобные молекулы, и способы ее применения
EP02770322A EP1440157B1 (fr) 2001-10-29 2002-10-29 Procedes et moyens de fabrication de proteines presentant des modifications post-traductionnelles predeterminees
ES02770322T ES2381104T3 (es) 2001-10-29 2002-10-29 Métodos y medios para producir proteínas con modificaciones postraduccionales predeterminadas
KR1020067008077A KR100737639B1 (ko) 2001-10-29 2002-10-29 예비결정된 번역후 변형을 갖는 단백질을 생성하기 위한장치 및 방법
US10/494,140 US7304031B2 (en) 2001-10-29 2002-10-29 Methods and means for producing proteins with predetermined post-translational modifications
IL16167402A IL161674A0 (en) 2001-10-29 2002-10-29 Methods and means for producing proteins with predetermined post-translational modifications
DK02770322.2T DK1440157T3 (da) 2001-10-29 2002-10-29 Methods and means for producing proteins with predetermined post-translational modifications
CN2007101494988A CN101177700B (zh) 2001-10-29 2002-10-29 生产具有预定的翻译后修饰的蛋白质的方法和手段
EA200602163A EA012340B1 (ru) 2001-10-29 2002-10-29 Способ получения гликозилированной белковой молекулы, содержащей структуру lewis x, и/или сиалил-lewis x, и/или lacdinac
EP10177590.6A EP2292770B1 (fr) 2001-10-29 2002-10-29 Procédés pour la production de protéines avec des modifications prédéterminées après translation
KR1020047006311A KR100692784B1 (ko) 2001-10-29 2002-10-29 예비결정된 번역후 변형을 갖는 단백질을 생성하기 위한장치 및 방법
CNB028216903A CN100347306C (zh) 2001-10-29 2002-10-29 生产具有预定的翻译后修饰的蛋白质的方法和手段
BR0213402-0A BR0213402A (pt) 2001-10-29 2002-10-29 Métodos para identificar uma célula de mamìfero capaz de produzir uma molécula proteinácea, para selecionar uma célula de mamìfero capaz de produzir uma molécula proteinácea, para se obter uma célula de mamìfero a partir de uma população heterogênea de células, e para produzir uma molécula proteinácea, composição farmaceuticamente aceitável, eritropoietina recombinantemente produzida, usos de uma célula de mamìfero, da eritropoietina recombinantemente produzida, e de uma composição de moléculas semelhantes à eritropoietina, preparação farmacêutica, método para o tratamento preventivo e/ou terapêutico de um distúrbio, composição de moléculas semelhates à eritropoietina, métodos para produzir, em uma célula de mamìfero, moléculas proteináceas, para produzir uma fração enriquecida em uma molécula proteinácea, e para fracionar uma mistura que contém moléculas proteináceas, fração, e, usos de uma fração ou composição, e de eritropoietina recombinantemente produzìvel em uma célula de mamìfero
AU2002335585A AU2002335585B2 (en) 2001-10-29 2002-10-29 Methods and means for producing proteins with predetermined post-translational modifications
JP2003540365A JP4583029B2 (ja) 2001-10-29 2002-10-29 所定の翻訳後修飾を有する蛋白質の製造方法及び製造手段
CA2756610A CA2756610C (fr) 2001-10-29 2002-10-29 Procedes de production de proteines comportant des glycanes n-lies comprenant des structures (sialyl-) lewis x ou lacdinac
IL161674A IL161674A (en) 2001-10-29 2004-04-29 Methods and means for producing proteins through predetermined post-translational modifications
NO20042209A NO20042209L (no) 2001-10-29 2004-05-28 Fremgangsmater og anordninger for fremstilling av proteiner med forutbestemte post-translasjonelle modifikasjoner
US11/102,073 US7297680B2 (en) 1999-04-15 2005-04-08 Compositions of erythropoietin isoforms comprising Lewis-X structures and high sialic acid content
US11/657,202 US7785833B2 (en) 2001-10-29 2007-01-24 Methods and means for producing proteins with predetermined post-translational modifications
US11/888,776 US7696157B2 (en) 2001-10-29 2007-08-01 Methods and means for producing proteins with predetermined post-translational modifications
IL201673A IL201673A (en) 2001-10-29 2009-10-21 A method for producing a portion of protein molecules enriched with glycans in n
IL201674A IL201674A (en) 2001-10-29 2009-10-21 Methods for producing proteins through predetermined post-translational modifications

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108888755A (zh) * 2018-07-11 2018-11-27 温州医科大学 红细胞生成素的新用途
CN110760476A (zh) * 2019-09-03 2020-02-07 广州瑞臻再生医学科技有限公司 一种大脑皮层神经干细胞及谷氨酸能神经元的制备方法

Citations (3)

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Publication number Priority date Publication date Assignee Title
US5856298A (en) * 1989-10-13 1999-01-05 Amgen Inc. Erythropoietin isoforms
WO2000061164A1 (fr) * 1999-04-13 2000-10-19 Kenneth S. Warren Laboratories Modulation de la fonction d'un tissu excitable par administration peripherique d'une erythropoietine
WO2000063403A2 (fr) * 1999-04-15 2000-10-26 Crucell Holland B.V. Production de proteine de recombinaison dans une cellule humaine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5856298A (en) * 1989-10-13 1999-01-05 Amgen Inc. Erythropoietin isoforms
WO2000061164A1 (fr) * 1999-04-13 2000-10-19 Kenneth S. Warren Laboratories Modulation de la fonction d'un tissu excitable par administration peripherique d'une erythropoietine
WO2000063403A2 (fr) * 1999-04-15 2000-10-26 Crucell Holland B.V. Production de proteine de recombinaison dans une cellule humaine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108888755A (zh) * 2018-07-11 2018-11-27 温州医科大学 红细胞生成素的新用途
CN110760476A (zh) * 2019-09-03 2020-02-07 广州瑞臻再生医学科技有限公司 一种大脑皮层神经干细胞及谷氨酸能神经元的制备方法

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