EP2167539A2 - Methods for producing glycosylated human alpha-1 antitrypsin (a1at) in mammalian host cells - Google Patents

Methods for producing glycosylated human alpha-1 antitrypsin (a1at) in mammalian host cells

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Publication number
EP2167539A2
EP2167539A2 EP08773472A EP08773472A EP2167539A2 EP 2167539 A2 EP2167539 A2 EP 2167539A2 EP 08773472 A EP08773472 A EP 08773472A EP 08773472 A EP08773472 A EP 08773472A EP 2167539 A2 EP2167539 A2 EP 2167539A2
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EP
European Patent Office
Prior art keywords
alat
glycosylation
antitrypsin
mammalian host
gene sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08773472A
Other languages
German (de)
French (fr)
Inventor
Markus Berger
Uwe Marx
Volker Sandig
René BRECHT
Susann Koch
Richard Ammer
Silke Rieck
Werner Reutter
Veronique Blanchard
Matthias Kaup
Rudolf Tauber
Stefan Zietze
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Charite Universitaetsmedizin Berlin
ProBioGen AG
Original Assignee
Charite Universitaetsmedizin Berlin
ProBioGen AG
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Filing date
Publication date
Application filed by Charite Universitaetsmedizin Berlin, ProBioGen AG filed Critical Charite Universitaetsmedizin Berlin
Publication of EP2167539A2 publication Critical patent/EP2167539A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin

Definitions

  • the present invention relates to methods for modulating the glycosylation status of human alpha-1 antitrypsin (AlAT) in mammalian host cells and methods for producing glycosylated human AlAT, to human alpha-1 antitrypsin obtained by these methods and to uses thereof.
  • the methods of the invention result in a AlAT that has a human glycosylation pattern or that has a modulated glycosylation pattern.
  • the present invention enables the production of AlAT with a "customized" glycosylation status which is suitable for different uses, preferably in human therapy.
  • Alpha-1 antitrypsin or ⁇ i -antitrypsin also known as serum trypsin inhibitor, is a serine protease inhibitor (serpin). It protects tissue from enzymes from inflammatory cells, especially elastase, and is present in human blood at 1.5 - 3.5 gram/liter.
  • AlAT Human alpha-1 antitrypsin
  • AlAT Human alpha-1 antitrypsin
  • Normal AlAT contains 394 residues, with three complex oligosaccharide units exposed to the surface of the molecule, linked to asparagines 46, 83, and 247 (Carrell et al., 1982).
  • AlAT is the major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of trypsin, elastase, and chymotrypsin in plasma.
  • the protein is a potent inhibitor of neutrophile elastase.
  • AlAT deficiency is one of the most common hereditary disorders. A deficiency of AlAT has been observed in a number of patients with chronic emphysema of the lungs. A proportion of individuals with serum deficiency of AlAT may progress to cirrhosis and liver failure (Wu et al., 1991).
  • AlAT As an elastase inhibitor, and because of the prevalence of genetic diseases resulting in deficient serum levels of AAT, there has been an active interest in recombinant synthesis of AAT, for human therapeutic use.
  • the AlAT produced by the above methods is either unglycosylated or has a glycosylation pattern different to that of human AlAT.
  • Chang et al. 2003 disclose the production of sialylated human AlAT in insect cells by introducing human/mammalian N-glycosyliation machinery into the insect cells.
  • Chang et al. describe, that because asialylated glycoproteins have a shorter half-life in blood circulation, it was investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells.
  • h ⁇ lAT human ⁇ l -antitrypsin
  • GnT2 GIcNAc transferase II
  • ⁇ l4GT ⁇ l,4-galactosyltransferase
  • ⁇ 26ST ⁇ 2,6-sialyltransferase
  • human AlAT can be produced in the milk of transgenic animals.
  • Archibald et al., 1990 examined the potential of transgenic animals as an alternative means of producing human AlAT.
  • a hybrid gene constructed by using sequences from an ovine milk protein fused to an AlAT "minigene" was used to generate transgenic mice.
  • Human AlAT was secreted into the milk of the mice.
  • AlAT from transgenic mouse milk was similar in size to human plasma-derived AlAT and showed a similar capacity to inhibit trypsin.
  • US 5,650,503 disclose genetic constructs comprising a 5' flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein, such as AlAT for producing proteins in transgenic animals.
  • WO 00/63403 A3 (Introgene B.V., Bout et al.) and US 2007/0054394 Al (Bout et al.) disclose a method for stably expressing human recombinant proteins, such as glycoproteins, in the human cell line PER.C6TM, which originates from embryonic retina cells (ECACC number 96022940). As an example, the production of human erythropoetin is described.
  • WO 99/61650, US 5,705,364 and US 2004/0259205 Al disclose processes for preparing glycoproteins by mammalian cell culture wherein the sialic acid content of the glycoprotein produced is controlled by manipulating the cell culture environment.
  • process parameters such as osmolality, temperature, addition of organic acids and salts thereof, transcription enhancers, addition of copper ions to the cell culture medium, on the cell specific productivity and the sialic acid content of the glycoprotein is described.
  • the production of TNFRl -IgG-subtyp 1 is shown.
  • WO 95/23233 disclose methods for modifying the carbohydrate moiety on glycoproteins to facilitate the structural and functional analysis of said glycoproteins, such as by NMR spectroscopic analysis and crystallography, which comprises treating glycoprotein-secreting mammalian cells having low endomannosidase activity under cell culture maintenance conditions with a glucosidase (I) inhibitor, and after secretion and purification, subsequent treatment of the active glycoprotein with endoglycosidase H to thereby provide a glycoprotein with a single GIcNAc residue at each glycosylation sequon.
  • the preferred mammalian cells are (CHO) cells and the preferred glucosidase (I) inhibitor is N-butyl deoxynojirimycin.
  • US 2005/0084933 Al disclose methods for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture.
  • the methods comprise feeding the cells with D-galactose, preferably with feed medium containing D-galactose, to sustain a sialylation effective level of D-galactose in the culture for its duration, thus increasing sialylation of the produced proteins.
  • the problem is solved by the present invention by providing a method by providing a method for modulating the glycosylation status of human alpha-1 antitrypsin (AlAT), i.e. a method for modulating the glycosylation of human alpha-1 antitrypsin (AlAT) .
  • AlAT human alpha-1 antitrypsin
  • Human alpha-1 antitrypsin (AlAT) according to the present invention is a protein encoded by a nucleic acid sequence as disclosed in Ciliberto et al., 1984. For detailed sequence information see e.g. Genbank accession number AAA51546 or Swiss-Prot Entry PO 1009, respectively. SEQ ID NO. 1 shows a nucleotide sequence of human AlAT and SEQ ID NO. 2 shows an amino acid sequence of human AlAT, see also Figures 2A and 2B. Furthermore, homologues of 90 %, preferably 99 % homology compared to the above nucleic acid sequence and or protein fall also within the meaning of "human alpha-1 antitrypsin” according to this invention.
  • the method for modulating the glycosylation status of human AlAT comprises culturing a mammalian host cell which expresses AlAT in cell culture under conditions that allow for protein production.
  • Cell culture conditions that allow for protein production are known in the art.
  • the method for modulating the glycosylation status of human alpha- 1 antitrypsin (AlAT) of the present invention is characterized in that the glycosylation of AlAT is modulated or controlled.
  • glycosylation is the process or result of addition of monosaccharides and/or saccharides to proteins and lipids. The process is one of four principal co-translational and post-translational modification steps in the synthesis of membrane and secreted proteins and the majority of proteins synthesized in the rough ER undergo glycosylation. Glycosylation represents the most common post-translational modification of proteins. It is an enzyme-directed site- specific process. Two main types of glycosylation exist: N-linked glycosylation to the amide nitrogen of asparagine side chains and O-linked glycosylation to the hydroxyl oxygen of serine and threonine side chains.
  • Glycans of glycoproteins are synthesized in the Golgi apparatus by specific glycosyltransferases, which attach nucleotide-activated monosaccharides to specific residues of glycoproteins.
  • the saccharide chains or glycans attached to the target proteins serve various functions. For instance, some proteins do not fold correctly unless they are glycosylated first.
  • glycans linked to a protein confer stability on some secreted glycoproteins.
  • the unglycosylated protein degrades quickly or is unprotected against the proteolytic activity of proteases. Glycosylation plays a role in cell- cell and cell-matrix signal transduction. Moreover, the glycosylation of glycoproteins is responsible for their biological activity, plasma half life and immunogenicity.
  • glycosylation There are two basic types of glycosylation which occur on asparagine (Asp, N) side chains, i.e. N-linked glycosylation, and serine (Ser, S) and threonine (Thr, T) side chains, i.e.O-linked glycosylation.
  • O-linked oligosaccharide chains of glycoproteins vary in complexity. They link to a protein via a glycosidic bond between a sugar residue and a serine or threonine hydroxyl group.
  • N- acetylglucosamine (abbreviated GIcNAc) is a common O-linked glycosylation of protein serine or threonine residues.
  • N-linked oligosaccharides of glycoproteins tend to be complex and branched.
  • N-acetylglucosamine is linked to a protein via the side-chain N of an asparagine residue in a particular 3-amino acid sequence (AsnSerThr, NST). Additional monosaccharides are added, and the N-linked oligosaccharide chain is modified by removal and addition of residues, to yield a characteristic branched structure.
  • ⁇ -glycosylation sites have an amino acid consensus sequence or recognition sequence, which is Asn-Xaa-Ser ( ⁇ XS) or Asn-Xaa-Thr ( ⁇ XT), wherein Xaa can be any amino acid, except Pro.
  • glycosylation on non naturally occurring amino acids is possible as well, such as hydroxyproline, hydroxylysine.
  • AlAT contains three glycosylation sites, which are ⁇ -glycosylation sites (hereinafter "original glycosylation sites”) which are
  • N83 N - Asn83 in the N 83 -domain LEG LNF N 83 LT EIP EAQ
  • N247 N 247 -domain LMK YLG N 247 AJ AIF FLP.
  • E 1 glutamic acid
  • N 46 domain, N 83 domain and/or N 247 domain are hereinafter referred to as "original glycosylation domains”.
  • AlAT furthermore comprises regions and sequence segments which are exposed at its surface and, thus, available to post-translational modification, such as glycosylation. Loops usually embrace secondary structures, such as alpha-helices and/or beta-sheets.
  • the crystal structure of AlAT has been disclosed, e.g. in Song et al., 1995 and Elliott et al., 2000.
  • loop regions The following five surface exposed or loop regions of AlAT have been chosen by the inventors and are hereinafter referred to as "loop regions":
  • loop A amino acids N83-H93
  • loop B amino acids Nl 04-Ql 11
  • loop C3 loop Cl (amino acids S121-V127)
  • loop C2 amino acids V145-H150
  • loop C3 amino acids Ql 66-Vl 81
  • loop D amino acids E199-V216
  • loop E amino acids D317-L327)
  • a "human glycosylation status or pattern" of AlAT means a glycosylation at the three original glycosylation sites and a glycosylation of a complex type, as e.g. described in Kolarich et al., 2006, which is incorporated herein by reference.
  • the three original iV-glycosylation sites of AlAT contain diantennary JV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
  • a "modulated glycosylation status or pattern" of AlAT means: a) a higher and/or different degree of glycosylation, (due to inserted and/or deleted glycosylation sites, as described in detail herein), b) an untypical high degree of a homogenous glycosylation, (e.g. lesser structure diversification or microheterogeneity of glycans) c) new sialic acids as terminal sugars
  • a "modulated glycosylation status or pattern" of AlAT is obtained by the methods of modulating the glycosylation status of AlAT according to the present invention, as described herein.
  • the modulation or control of glycosylation of AlAT comprises preferably one or more of the following: a) modifying the AlAT gene sequence and/or b) modifying the glycosylation machinery of the mammalian host cell and/or c) varying the cell culture conditions.
  • one of the advantages of the method for modulating the glycosylation status of AlAT according to the present invention is that the glycosylation status can be adjusted dependent on the purpose of the AlAT to be produced. "Customized" glycosylation patterns can be obtained (see below for more details).
  • the problem is furthermore solved by providing a method for producing glycosylated human alpha-1 antitrypsin (AlAT) in a mammalian host cell, further comprising the following steps: i) providing a mammalian host cell, ii) providing a nucleotide sequence comprising the AlAT gene sequence, iii) transfecting said mammalian host cell with the nucleotide sequence comprising the
  • AlAT gene sequence iv) culturing the transfected mammalian host cell, v) obtaining AlAT expressed by the cultured mammalian host cell.
  • This producing method is as well characterized in that the glycosylation of AlAT is modulated or controlled.
  • Said modulation or control of glycosylation of AlAT according to the present invention comprises preferably one or more of the following: a) modifying the AlAT gene sequence and/or b) modifying the glycosylation machinery of the mammalian host cell and/or c) varying the cell culture conditions.
  • modifying the AlAT gene sequence comprises one or more of the following:
  • modifying the AlAT gene sequence comprises the insertion and/or deletion of one ore more glycosylation sites into the AlAT gene sequence.
  • the glycosylation sites are N-glycosylation sites.
  • glycosylation sites, that are inserted into the AlAT gene sequence are inserted at one or more positions that are different to the original glycosylation sites within the AlAT gene sequence.
  • positions that are different to the original glycosylation sites within the AlAT gene sequence are preferably selected from positions in the loop regions, preferably loop A, loop B, loop C, loop D and loop E, as defined above. These positions can preferably be identified using a mutation strategy. Further positions can be identified by a person of skill in the art by applying the teaching of the present invention.
  • glycosylation sites are inserted and/or deleted at one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence.
  • the one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence are preferably selected from one or more of the following: N 46 domain, N 83 domain and N 247 domain. These positions can preferably be identified using a mutation strategy. Further positions can be identified by a person of skill in the art by applying the teaching of the present invention.
  • one or more original glycosylation sites within the AlAT gene sequence are deleted, wherein the original glycosylation sites are selected from N46, N83 and N247. These positions can preferably be identified using a mutation strategy. In some embodiments this deletion can occur while glycosylation sites are inserted, see below.
  • the one or more glycosylation site that is inserted is selected from the group comprising N48, N81, N90, N 108, N 123, N201, N249 and N323.
  • the one or more glycosylation site that is inserted resembles or introduces the consensus or recognition sequence for a N-glycosylation site (as described above), thus the one or more glycosylation site that is inserted is more preferably selected from the group comprising N48/T50, N81/T83, N90/T92, N108/T110, N123/T125, N201, N249/T251 and
  • the following primers can be used for introducing these glycosylation sites into AlAT:
  • modifying the AlAT gene sequence comprises the insertion of a glycosylation tag.
  • the glycosylation tag comprises one or more glycosylation sites or one or more glycosylation domains of another glycoprotein or the glycosylation tag comprises one or more synthetic glycosylation domains, i.e. not based on a known glycoprotein.
  • the resulting protein is a chimera of (a) AlAT or parts of it and at least one of the follwing
  • the other glycoprotein is a highly glycosylated glycoprotein, preferably a glycoprotein with highly glycosylated glycosylation domain(s).
  • Preferred highly glycosylated glycoproteins are alphal-acid glycoprotein (AGP) (Higai et al., 2005) and erythropoietin (EPO) (Takeuchi et al., 1991).
  • modifying the glycosylation machinery of the mammalian host cell comprises one or more of the following: a) knock out of specific glycosyltransferases, such as fucosyltransferases, b) loss-of function mutation of the epimerase/ManNAc kinase for producing a sialuria mutant, c) simultaneous co-transfection of one or several glycosidases, and/or d) knock out of specific glycosidase genes.
  • the "glycosylation machinery" of a mammalian host cell according to the present invention comprises all enzymes which take part in building up the different types of glycosylation (e.g. N-, O-glycans), such as glycosyltransferases, epimerases, ManNAc kinase, sialyltransferases, synthases.
  • the "glycosylation machinery" of a mammalian host cell according to the present invention comprises furthermore the enzymes which are responsible for the rearrangement, degradation and recycling of the glycans.
  • varying the cell culture conditions comprises the addition of sugars or sugar derivatives to the cell culture medium.
  • the sugars or sugar derivatives are selected from N-acetyl mannosamine and derivatives thereof, e.g. 2-desoxy-2-N-propanoylamino-D-mannose (ManNProp), 2-desoxy-2- N-Cyclopropyl-acetyl- amino-D-mannose (ManNcyProp), 2-desoxy-2-N-pentanoylamino-D- mannose (ManNPent) (see also Figures 3 and 4).
  • ManNProp 2-desoxy-2-N-propanoylamino-D-mannose
  • ManNcyProp 2-desoxy-2- N-Cyclopropyl-acetyl- amino-D-mannose
  • ManNPent 2-desoxy-2-N-pentanoylamino-D- mannose
  • Sialylation of glycoco ⁇ jugates is essential for mammalian cells.
  • Sialic acid is synthesized in the cytosol from N-acetyl mannosamine by several consecutive steps.
  • N-propanoyl mannosamine a novel precursor of sialic acid, it is possible to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoco ⁇ jugates taking advance of the cellular sialylation machinery.
  • N-propanoylneuraminic acid One terminal monosaccharide of nearly all glycoproteins.
  • One terminal monosaccharide of nearly all glycoproteins is sialic acid.
  • the most frequent sialic acid in humans is iV-acetylneuraminic acid (Neu5 Ac).
  • the physiological precursor of all sialic acids is iV-acetyl D-mannosamine (ManNAc).
  • Synthetic N-acyl-modified D- mannosamines such as iV-propanoyl D-mannosamine (ManNProp) can be taken up by cells and efficiently metabolized to the respective iV-acyl-modified sialic acid in vitro and in vivo which can then be introduced into cellular glycoconjugates (see Figure 3).
  • varying the cell culture conditions furthermore comprises the variation of one or more of the following parameters:
  • the following cell culture conditions are preferably varied depending on the cultivation mode
  • the bioreactor system used in the perfusion process is a liquid / gas phase exposure bioreactor family for cell cultivation as disclosed in WO2005/121311A1 and WO 2006/120202A1 (ProBioGen AG) 5 which are herein incorporated by reference in their entirety. See also Langhammer et al., 2007 (ProBioGen) which is enclosed herewith by reference.
  • a cell culture device and method for growing cells and for cell cultivation in high density whereby the cells for cultivation are located in hollow fibre membranes and are alternately supported in a liquid nutrient and a gas phase thereabove.
  • the device is a liquid/gas phase exposure bioreactor with a supply chamber, in which hollow fibre membranes with an inner diameter of no more than 5 mm are located and the inner volumes of which form culture chambers. After introduction of the cells into the culture chambers approximately half of the supply chamber is filled with nutrient medium and the other half with a gas mixture. After switching on the medium and gas perfusion, a cyclic exposure of the hollow fibre membranes and the cells therein to the gas or the liquid phase begins.
  • WO2005/12131 IAl (ProBioGen) for more details see WO2005/12131 IAl (ProBioGen).
  • a device for growing and cultivating cells in membrane- based bioreactors which have a design that represents a scaling down of corresponding production bioreactors and which can be individually regulated with regard to temperature, pH, gas supply, mixing regime and nutrient supply.
  • a device is characterised in that preferably at least six membrane-based bioreactors can be simultaneously operated in a device having a single energy supply and a centralised control and regulation unit.
  • the mammalian host cell that is used in the methods of the present invention is a potent expression cell line, which has high synthetic capacity, such as high expression yields, and an extended cell viability. It is preferred that the mammalian host cell that is used in the methods of the present invention is a human neuronal cell, preferably NC5T11 and relatives thereof.
  • NC5T11 and relatives thereof are disclosed in WO 2007/05416 Al (ProBioGen AG), which is herein incorporated by reference in its entirety.
  • a mammalian host cell suitable for the methods of the present invention is a human cell, a rodent cell including mouse, rat, hamster cell, etc., more preferably is a human brain derived cell including human foetal brain cells such as foetal neurons and foetal glia cells.
  • Preferred mammalian host cells suitable used for the methods of the present invention are high expression cell lines having stably integrated into their genome a gene encoding a specific heterologous regulator protein or a functional variant thereof and stably expressing said regulator protein or the functional variant thereof.
  • the heterologous regulator protein preferably modulates transcription and/or cell growth, and enhances the productivity of the cell in the production of a protein differing from said regulator protein or the functional variant thereof, such as AlAT.
  • the functional variant of the heterologous regulator protein is preferably a fusion protein, preferably said fusion protein comprising at least one first domain comprising an adenovirus PIX regulator protein and at least one second domain that modulates or expands the activity or the subcellular distribution of the adenovirus pIX; and/or said at least one second domain comprises a protein or peptide acting as a transcription modulator, preferably said transcription modulator is a transcription factor including the retinoic acid receptor alpha, is a marker protein, preferably said marker protein is a fluorescence marker including GFP, DsRed and its variants or is an enzyme including LacZ, or is a transit peptide including a NLS.
  • heterologous regulator protein is the adenovirus PIX, or (ii) is a fusion protein between PIX and retinoic acid receptor alpha, or (iii) is a fusion protein between PIX 5 and GFP and containing an NLS sequence, or (iv) is a fusion protein between PIX and GFP or between PIX and an isolated NLS sequence.
  • mammalian host cells suitable used for the methods of the present invention preferably NC5T11 and relatives, carries further immortalizing (viral) genes including an El protein of an adenovirus, preferably of mastadenovirus group C type 5, most preferably the cell carries the adenovirus ElA and/or ElB gene; and/or (iv) further carries functional sequences such as selection marker sequences, splice donor/acceptor sites and/or recombinase recognition sequences allowing integration of a target nucleic acid sequence to be expressed in the cell.
  • immortalizing (viral) genes including an El protein of an adenovirus, preferably of mastadenovirus group C type 5, most preferably the cell carries the adenovirus ElA and/or ElB gene; and/or (iv) further carries functional sequences such as selection marker sequences, splice donor/acceptor sites and/or recombinase recognition sequences allowing integration of a target nucleic acid sequence to be expressed in
  • the problem is furthermore solved by the present invention by providing glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • the glycosylation status or pattern of the human alpha- 1 antitrypsin (AlAT) obtained is modulated compared to wild type human AlAT.
  • a "human glycosylation status or pattern" of AlAT means a glycosylation at the three original glycosylation sites and a glycosylation of a complex type, as e.g. described in Kolarich et al., 2006, which is incorporated herein by reference.
  • the three original iV-glycosylation sites of AlAT contain diantennary iV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
  • a "modulated glycosylation status or pattern" of Al AT according to the present invention means: a) a higher and/or different degree of glycosylation,
  • a "modulated glycosylation status or pattern" of AlAT is obtained by the methods of modulating the glycosylation status of AlAT according to the present invention, as described herein.
  • the human alpha- 1 antitrypsin (AlAT) obtained by the methods of this invention and which is characterized by a modulated glycosylation status or pattern is advantageous for use in treating AlAT deficiency e.g. due to an increased effective serum half life and reduced biological side effects.
  • the problem is furthermore solved by the present invention by using the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above, for the treatment of diseases, preferably human therapy.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • the disease to be treated is selected from the group of AlAT deficiency, deficiency emphysema, neonatal cholestasis, chronic hepatic cirrhosis and cystic fibrosis.
  • the disease is AlAT deficiency.
  • the person of skill in the art will be able to determine further uses of AlAT for treating and/or preventing diseases and for human therapy.
  • the problem is furthermore solved by the present invention by providing a method of treatment of a disease, wherein the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above is applied to a patient in need thereof.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • the disease to be treated is selected from the group of AlAT deficiency, deficiency emphysema, neonatal cholestasis, chronic hepatic cirrhosis and cystic fibrosis.
  • the disease is AlAT deficiency.
  • the person of skill in the art will be able to determine further treatment and/or prevention methods of diseases and for human therapy.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • the problem is furthermore solved by the present invention by providing a diagnosis method, wherein the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above is used as a diagnosis standard for in vitro and or in vivo measuring the plasma life time, clearance, concentration and other parameters of AlAT, preferably recombinant AlAT.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • the problem is furthermore solved by the present invention by providing a kit, preferably a diagnosis kit comprising the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention.
  • a kit preferably a diagnosis kit comprising the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention.
  • AlAT glycosylated human alpha- 1 antitrypsin
  • FIG. 1 A ribbon diagram of the structure of alpha- 1 antitrypsin in complex with trypsin is shown, wherein AlAT is cleaved and wherein the three original glycosylation sites N46, N83 and N247 of AlAT are shown as well as loops A, B, C, D and E, which exemplify preferred sites for inserting additional glycosylation sites according to the present invention.
  • Figure 2A and 2B Sequence of AlAT.
  • the DNA sequence (SEQ ID NO. 1) and the amino acid sequence of AlAT (SEQ ID NO. 2) are shown, including the signal peptide, numbering starts at Glul (El).
  • the loop regions are underlined.
  • Loop C is made of 3 parts, i.e. loop Cl, loop C2 and loop C3.
  • N* original N- glycosylation sites (N46, N83, N247).
  • mutations and the respective eight primer sequences are shown.
  • FIG. 3 Schematic representation of the incorporation of unnatural sialic acids into glycoproteins involving ManNProp (2-desoxy-2-N-propanoylamino-D-mannose). Note that sialic acids are activated in the nucleus to the respective CMP-activated sialic acid. Activated sialic acids are then transported across the cytosol into the golgi compartment, where specific sialiyltransferases are located.
  • Figure 5 Batch process to produce AlAT by expression in human neuronal cells. Cell count and viability in the AlAT production process with NC5T11 (also called AGEl. hn) in cellfern (pro are shown. The cells grow well with a stable viability over a period of more than 9 days.
  • NC5T11 also called AGEl. hn
  • pro pro are shown. The cells grow well with a stable viability over a period of more than 9 days.
  • FIG. Perfusion process to produce AlAT by expression in human neuronal cells. Glucose consumption and protein production per bioreactor module are shown.
  • Mode of cell culture There are two modes for culturing the mammalian host cells:
  • Perfusion process in a membrane based bioreactor cells are grown inside the membrane chambers of the bioreactor-module.
  • the space surrounding the membranes is perfused continuously with medium and gas.
  • the supply of the cells inside the membrane chambers is ensured by mass diffusion through the membrane.
  • the AlAT can pass the membrane from inside to the extracapillary space whereas the cells are retained by the membrane. By this way the AlAT is continuously harvested with the medium perfusion flow and can be stored coolly fast.
  • the bioreactor system used in the perfusion process (b) is a liquid / gas phase exposure bioreactor family for cell cultivation as disclosed in WO2005/121311A1 and WO 2006/120202A1 (ProBioGen AG), which are herein incorporated by reference in their entirety.
  • NC5T11 also called AGEl.hn cells were cultivated in batch form in cellferm (pro) (DasGip AG, Julich, Germany). Start volume was 450ml. Cell count, viability and AlAT- concentration were measured every second day. In Figure 5 cell count and viability are shown. The cells grow well with a stable viability.
  • NC5T11 also called AGEl.hn cells were cultivated in a process development device of a perfusion-bioreactor for 31 days. Start cell count was 1.5 x 10 vital cells absolute per module. The flow rate was 25ml/d. Glucose consumption and protein production per day and module were measured/ calculated (see Figure 6).

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Abstract

The present invention relates to methods for modulating the glycosylation status of human alpha-1 antitrypsin (A1AT) in mammalian host cells and methods for producing glycosylated human A1AT, to human alpha-1 antitrypsin obtained by these methods and to uses thereof. The methods of the invention result in a A1AT that has a human glycosylation pattern or that has a modulated glycosylation pattern. Thus, the present invention enables the production of A1AT with a "customized" glycosylation status which is suitable for different uses, preferably in human therapy.

Description

Methods for producing glycosylated human alpha-1 antitrypsin (AlAT) in mammalian host cells
The present invention relates to methods for modulating the glycosylation status of human alpha-1 antitrypsin (AlAT) in mammalian host cells and methods for producing glycosylated human AlAT, to human alpha-1 antitrypsin obtained by these methods and to uses thereof. The methods of the invention result in a AlAT that has a human glycosylation pattern or that has a modulated glycosylation pattern. Thus, the present invention enables the production of AlAT with a "customized" glycosylation status which is suitable for different uses, preferably in human therapy.
BACKGROUND OF THE INVENTION
Alpha-1 antitrypsin or αi -antitrypsin (AlAT), also known as serum trypsin inhibitor, is a serine protease inhibitor (serpin). It protects tissue from enzymes from inflammatory cells, especially elastase, and is present in human blood at 1.5 - 3.5 gram/liter.
Human alpha-1 antitrypsin (AlAT) is a monomer with a molecular weight of about 52 kD. Normal AlAT contains 394 residues, with three complex oligosaccharide units exposed to the surface of the molecule, linked to asparagines 46, 83, and 247 (Carrell et al., 1982). AlAT is the major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of trypsin, elastase, and chymotrypsin in plasma. In particular, the protein is a potent inhibitor of neutrophile elastase.
AlAT deficiency is one of the most common hereditary disorders. A deficiency of AlAT has been observed in a number of patients with chronic emphysema of the lungs. A proportion of individuals with serum deficiency of AlAT may progress to cirrhosis and liver failure (Wu et al., 1991).
To date, the most common therapy of AlAT deficiency is the administration of human AlAT isolated from plasma fractions. However, new therapeutic approaches, such as administration of recombinant AlAT, gene therapy as well as administration of synthetic elastase inhibitors are investigated.
Because of the key role of AlAT as an elastase inhibitor, and because of the prevalence of genetic diseases resulting in deficient serum levels of AAT, there has been an active interest in recombinant synthesis of AAT, for human therapeutic use.
Expression of AlAT
- in non mammalian cells
There are a number of approaches for expressing mammalian/human AlAT in microorganisms, such as bacteria (EP 0 137 633 Al of ZymoGenetics) or yeast (US 4,839,283, EP 0 304 971 A2 of ZymoGenetics or US 4,752,576 of Chiron Corporation).
Furthermore, the expression of AlAT in plants has been reported, see e.g. US 6,127,145 or Sudarshana et al., 2006.
However, the AlAT produced by the above methods is either unglycosylated or has a glycosylation pattern different to that of human AlAT.
Chang et al. 2003, disclose the production of sialylated human AlAT in insect cells by introducing human/mammalian N-glycosyliation machinery into the insect cells. Chang et al. describe, that because asialylated glycoproteins have a shorter half-life in blood circulation, it was investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. In two insect cell lines, Sf9 and Ea4, the human αl -antitrypsin (hαlAT) protein was co-expressed with a series of key glycosyltransferases, including GIcNAc transferase II (GnT2), βl,4-galactosyltransferase (βl4GT), and α2,6-sialyltransferase (α26ST) by a single recombinant baculovirus.
- in mammalian cells
US 5,399,684 and US 5,736,379 (Washington Research Foundation) disclose the DNA sequences of mammalian AlAT which can be used for the expression of AlAT in mammalian cells. The expression of human AlAT in heterologous mammalian cells is known and described in several publications, such as for CHO cells in Paterson et al. (1994).
- in transgenic animals
Alternatively to producing AlAT in mammalian cell culture, human AlAT can be produced in the milk of transgenic animals. Archibald et al., 1990 examined the potential of transgenic animals as an alternative means of producing human AlAT. A hybrid gene constructed by using sequences from an ovine milk protein fused to an AlAT "minigene" was used to generate transgenic mice. Human AlAT was secreted into the milk of the mice. AlAT from transgenic mouse milk was similar in size to human plasma-derived AlAT and showed a similar capacity to inhibit trypsin.
US 5,650,503 (PPL Therapeutics) disclose genetic constructs comprising a 5' flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein, such as AlAT for producing proteins in transgenic animals.
Expression of glycoproteins in mammalian cells
WO 00/63403 A3 (Introgene B.V., Bout et al.) and US 2007/0054394 Al (Bout et al.) disclose a method for stably expressing human recombinant proteins, such as glycoproteins, in the human cell line PER.C6™, which originates from embryonic retina cells (ECACC number 96022940). As an example, the production of human erythropoetin is described.
WO 99/61650, US 5,705,364 and US 2004/0259205 Al (all of Genentech) disclose processes for preparing glycoproteins by mammalian cell culture wherein the sialic acid content of the glycoprotein produced is controlled by manipulating the cell culture environment. The influence of different process parameters, such as osmolality, temperature, addition of organic acids and salts thereof, transcription enhancers, addition of copper ions to the cell culture medium, on the cell specific productivity and the sialic acid content of the glycoprotein is described. The production of TNFRl -IgG-subtyp 1 is shown.
WO 95/23233 disclose methods for modifying the carbohydrate moiety on glycoproteins to facilitate the structural and functional analysis of said glycoproteins, such as by NMR spectroscopic analysis and crystallography, which comprises treating glycoprotein-secreting mammalian cells having low endomannosidase activity under cell culture maintenance conditions with a glucosidase (I) inhibitor, and after secretion and purification, subsequent treatment of the active glycoprotein with endoglycosidase H to thereby provide a glycoprotein with a single GIcNAc residue at each glycosylation sequon. The preferred mammalian cells are (CHO) cells and the preferred glucosidase (I) inhibitor is N-butyl deoxynojirimycin.
US 2005/0084933 Al (Bristol-Myers Squibb) disclose methods for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture. The methods comprise feeding the cells with D-galactose, preferably with feed medium containing D-galactose, to sustain a sialylation effective level of D-galactose in the culture for its duration, thus increasing sialylation of the produced proteins.
However, the above described approaches have not been satisfactory for the production of AlAT, because AlAT produced by microbial recombinant methods result in a recombinant protein with a short effective serum half life. The prior art methods for making AlAT using mammalian cell culture or transgenic animals, while expected to produce a more active AlAT, by virtue of a more human glycosylation pattern, are relatively expensive, and to date, have limited the available supply of glycosylated AAT.
Thus, there is a need in the art for methods and means for an improved production of glycosylated, therapeutically effective AlAT, which allows for inexpensive and/or customized production.
The problem is solved by the present invention by providing a method by providing a method for modulating the glycosylation status of human alpha-1 antitrypsin (AlAT), i.e. a method for modulating the glycosylation of human alpha-1 antitrypsin (AlAT) .
"Human alpha-1 antitrypsin" (AlAT) according to the present invention is a protein encoded by a nucleic acid sequence as disclosed in Ciliberto et al., 1984. For detailed sequence information see e.g. Genbank accession number AAA51546 or Swiss-Prot Entry PO 1009, respectively. SEQ ID NO. 1 shows a nucleotide sequence of human AlAT and SEQ ID NO. 2 shows an amino acid sequence of human AlAT, see also Figures 2A and 2B. Furthermore, homologues of 90 %, preferably 99 % homology compared to the above nucleic acid sequence and or protein fall also within the meaning of "human alpha-1 antitrypsin" according to this invention. The method for modulating the glycosylation status of human AlAT according to the present invention comprises culturing a mammalian host cell which expresses AlAT in cell culture under conditions that allow for protein production. Cell culture conditions that allow for protein production are known in the art.
The method for modulating the glycosylation status of human alpha- 1 antitrypsin (AlAT) of the present invention is characterized in that the glycosylation of AlAT is modulated or controlled.
"Glycosylation" is the process or result of addition of monosaccharides and/or saccharides to proteins and lipids. The process is one of four principal co-translational and post-translational modification steps in the synthesis of membrane and secreted proteins and the majority of proteins synthesized in the rough ER undergo glycosylation. Glycosylation represents the most common post-translational modification of proteins. It is an enzyme-directed site- specific process. Two main types of glycosylation exist: N-linked glycosylation to the amide nitrogen of asparagine side chains and O-linked glycosylation to the hydroxyl oxygen of serine and threonine side chains. Glycans of glycoproteins are synthesized in the Golgi apparatus by specific glycosyltransferases, which attach nucleotide-activated monosaccharides to specific residues of glycoproteins. The saccharide chains or glycans attached to the target proteins serve various functions. For instance, some proteins do not fold correctly unless they are glycosylated first. Also, glycans linked to a protein confer stability on some secreted glycoproteins. Furthermore, the unglycosylated protein degrades quickly or is unprotected against the proteolytic activity of proteases. Glycosylation plays a role in cell- cell and cell-matrix signal transduction. Moreover, the glycosylation of glycoproteins is responsible for their biological activity, plasma half life and immunogenicity.
There are two basic types of glycosylation which occur on asparagine (Asp, N) side chains, i.e. N-linked glycosylation, and serine (Ser, S) and threonine (Thr, T) side chains, i.e.O-linked glycosylation.
O-linked oligosaccharide chains of glycoproteins vary in complexity. They link to a protein via a glycosidic bond between a sugar residue and a serine or threonine hydroxyl group. N- acetylglucosamine (abbreviated GIcNAc) is a common O-linked glycosylation of protein serine or threonine residues. N-linked oligosaccharides of glycoproteins tend to be complex and branched. Initally, N-acetylglucosamine is linked to a protein via the side-chain N of an asparagine residue in a particular 3-amino acid sequence (AsnSerThr, NST). Additional monosaccharides are added, and the N-linked oligosaccharide chain is modified by removal and addition of residues, to yield a characteristic branched structure.
Ν-glycosylation sites have an amino acid consensus sequence or recognition sequence, which is Asn-Xaa-Ser (ΝXS) or Asn-Xaa-Thr (ΝXT), wherein Xaa can be any amino acid, except Pro.
However, glycosylation on non naturally occurring amino acids is possible as well, such as hydroxyproline, hydroxylysine.
AlAT contains three glycosylation sites, which are Ν-glycosylation sites (hereinafter "original glycosylation sites") which are
- Asn46 (hereinafter "Ν46") in the N46-domain QLA HQS N46SJ NIF FSP,
- Asn83 (hereinafter "N83") in the N83-domain LEG LNF N83LT EIP EAQ,
- Asn247 (hereinafter "N247") in the N247-domain LMK YLG N247AJ AIF FLP. (After cleavage of the signal peptide (23 aa) the numbering of the first amino acid begins with the glutamic acid: E1).
The N46 domain, N83 domain and/or N247 domain are hereinafter referred to as "original glycosylation domains".
A description of the glycosylation of human AlAT is given by Kolarich et al., 2006, which is incorporated herein by reference. The three N-glycosylation sites of AlAT contain diantennary iV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
AlAT furthermore comprises regions and sequence segments which are exposed at its surface and, thus, available to post-translational modification, such as glycosylation. Loops usually embrace secondary structures, such as alpha-helices and/or beta-sheets. The crystal structure of AlAT has been disclosed, e.g. in Song et al., 1995 and Elliott et al., 2000.
The following five surface exposed or loop regions of AlAT have been chosen by the inventors and are hereinafter referred to as "loop regions":
- loop A (amino acids N83-H93) (hereinafter "loop A") which overlaps with the N83-domain N83LT EIP EAQ IH,
- loop B (amino acids Nl 04-Ql 11) (hereinafter "loop B")
NQP DSQ LQ,
- loop C containing 3 parts, namely loop Cl (amino acids S121-V127) (hereinafter "loop C3")
SEG LKL V loop C2 (amino acids V145-H150) (hereinafter "loop C2")
VNF GDH and loop C3 (amino acids Ql 66-Vl 81) (hereinafter "loop C3")
QGK IVD LVK ELD RDT V (all 3 loops hereinafter ,,loop C"),
- loop D (amino acids E199-V216) (hereinafter "loop D")
EVK DTE DED FHV DQV TTV,
- loop E (amino acids D317-L327) (hereinafter "loop E")
LSG VTE EAP L. See also Figure 1 as well as Figures 2 A and 2B.
A "human glycosylation status or pattern" of AlAT according to the present invention means a glycosylation at the three original glycosylation sites and a glycosylation of a complex type, as e.g. described in Kolarich et al., 2006, which is incorporated herein by reference. The three original iV-glycosylation sites of AlAT contain diantennary JV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
A "modulated glycosylation status or pattern" of AlAT according to the present invention means: a) a higher and/or different degree of glycosylation, (due to inserted and/or deleted glycosylation sites, as described in detail herein), b) an untypical high degree of a homogenous glycosylation, (e.g. lesser structure diversification or microheterogeneity of glycans) c) new sialic acids as terminal sugars
(due to addition of sugars to the cell culture medium e.g. Neu5Prop instead of Neu5Ac, as described herein).
A "modulated glycosylation status or pattern" of AlAT is obtained by the methods of modulating the glycosylation status of AlAT according to the present invention, as described herein.
The modulation or control of glycosylation of AlAT according to the present invention comprises preferably one or more of the following: a) modifying the AlAT gene sequence and/or b) modifying the glycosylation machinery of the mammalian host cell and/or c) varying the cell culture conditions.
Thus, one of the advantages of the method for modulating the glycosylation status of AlAT according to the present invention is that the glycosylation status can be adjusted dependent on the purpose of the AlAT to be produced. "Customized" glycosylation patterns can be obtained (see below for more details).
The problem is furthermore solved by providing a method for producing glycosylated human alpha-1 antitrypsin (AlAT) in a mammalian host cell, further comprising the following steps: i) providing a mammalian host cell, ii) providing a nucleotide sequence comprising the AlAT gene sequence, iii) transfecting said mammalian host cell with the nucleotide sequence comprising the
AlAT gene sequence, iv) culturing the transfected mammalian host cell, v) obtaining AlAT expressed by the cultured mammalian host cell.
This producing method is as well characterized in that the glycosylation of AlAT is modulated or controlled. Said modulation or control of glycosylation of AlAT according to the present invention comprises preferably one or more of the following: a) modifying the AlAT gene sequence and/or b) modifying the glycosylation machinery of the mammalian host cell and/or c) varying the cell culture conditions.
Modifying the AlAT gene sequence
According to the present invention "modifying the AlAT gene sequence" comprises one or more of the following:
- the insertion of one ore more glycosylation sites into the AlAT gene sequence,
- the deletion of one ore more glycosylation sites into the AlAT gene sequence,
- the insertion of a glycosylation tag.
a) Insertion and/or deletion of one ore more glycosylation sites into the AlAT gene sequence It is preferred, that modifying the AlAT gene sequence comprises the insertion and/or deletion of one ore more glycosylation sites into the AlAT gene sequence.
Preferably, the glycosylation sites are N-glycosylation sites.
It is preferred, that the glycosylation sites, that are inserted into the AlAT gene sequence, are inserted at one or more positions that are different to the original glycosylation sites within the AlAT gene sequence.
The positions that are different to the original glycosylation sites within the AlAT gene sequence are preferably selected from positions in the loop regions, preferably loop A, loop B, loop C, loop D and loop E, as defined above. These positions can preferably be identified using a mutation strategy. Further positions can be identified by a person of skill in the art by applying the teaching of the present invention.
In a further embodiment of the present invention the glycosylation sites are inserted and/or deleted at one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence.
The one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence are preferably selected from one or more of the following: N46 domain, N83 domain and N247 domain. These positions can preferably be identified using a mutation strategy. Further positions can be identified by a person of skill in the art by applying the teaching of the present invention.
In a further embodiment of the present invention one or more original glycosylation sites within the AlAT gene sequence are deleted, wherein the original glycosylation sites are selected from N46, N83 and N247. These positions can preferably be identified using a mutation strategy. In some embodiments this deletion can occur while glycosylation sites are inserted, see below.
In a preferred embodiment, the one or more glycosylation site that is inserted is selected from the group comprising N48, N81, N90, N 108, N 123, N201, N249 and N323.
More preferably, the one or more glycosylation site that is inserted resembles or introduces the consensus or recognition sequence for a N-glycosylation site (as described above), thus the one or more glycosylation site that is inserted is more preferably selected from the group comprising N48/T50, N81/T83, N90/T92, N108/T110, N123/T125, N201, N249/T251 and
N323/T325.
For more detail please see Figure 2.
N-glycosylation site location influence on inserted into AlAT original glycosylation site
N48/T50 N46 domain deletion of N46
N81/T83 N83 domain deletion of N83
N90/T92 loop A
N108/T110 loop B
N123/T125 loop Cl
N201 loop D
N249/T251 N247 domain deletion of N247
N323/T325 loop E
The following primers can be used for introducing these glycosylation sites into AlAT:
Primer 1 N48, T50 SEQ ID NO. 3 ccagtccaacagcaAcaataCcttcttctcccc Primer 2 N81 , T83 SEQ ID NO. 4 gggcctgaat.ttcaCcctcacggagatt Primer 3 N90, T92 SEQ ID NO. 5 cggagAAtcagaCccatgaaggcttc
Primer 4 N108. T110 SEQ ID NO. 6 ccagccagacaAccagACccagctgaccacc
Primer 5 N123, T125 SEQ ID NO. 7 gttcctcagcgagAAcctgaCgctagtggataag
Primer 6 N201 SEQ ID NO. 8 tttgaagtcaaTgacaccgaggacgag
Primer 7 N249, T251 SEQ ID NO. 9 cctgggcaatgccaAcgccaCcttcttcctacc
Primer 8 N323, T325 SEQ ID NO. 10 ctccggggtcacaAaCgagAcacccctgaagctctc
The above described modifications of the AlAT gene sequence can be combined.
b) Insertion of a glycosylation tag
It is furthermore preferred, that modifying the AlAT gene sequence comprises the insertion of a glycosylation tag.
Preferably, the glycosylation tag comprises one or more glycosylation sites or one or more glycosylation domains of another glycoprotein or the glycosylation tag comprises one or more synthetic glycosylation domains, i.e. not based on a known glycoprotein.
Thus, the resulting protein is a chimera of (a) AlAT or parts of it and at least one of the follwing
(b) one or more glycosylation sites of another glycoprotein or
(c) one or more glycosylation domains of another glycoprotein or
(d) one or more synthetic glycosylation domains or
(e) one or more glycosylation sites of a synthetic glycosylation domain.
It is preferred, that the other glycoprotein is a highly glycosylated glycoprotein, preferably a glycoprotein with highly glycosylated glycosylation domain(s). Preferred highly glycosylated glycoproteins are alphal-acid glycoprotein (AGP) (Higai et al., 2005) and erythropoietin (EPO) (Takeuchi et al., 1991).
Modifying the glycosylation machinery of the mammalian host cell
According to the present invention modifying the glycosylation machinery of the mammalian host cell comprises one or more of the following: a) knock out of specific glycosyltransferases, such as fucosyltransferases, b) loss-of function mutation of the epimerase/ManNAc kinase for producing a sialuria mutant, c) simultaneous co-transfection of one or several glycosidases, and/or d) knock out of specific glycosidase genes.
The "glycosylation machinery" of a mammalian host cell according to the present invention comprises all enzymes which take part in building up the different types of glycosylation (e.g. N-, O-glycans), such as glycosyltransferases, epimerases, ManNAc kinase, sialyltransferases, synthases. The "glycosylation machinery" of a mammalian host cell according to the present invention comprises furthermore the enzymes which are responsible for the rearrangement, degradation and recycling of the glycans.
Varying the cell culture conditions
According to the present invention varying the cell culture conditions comprises the addition of sugars or sugar derivatives to the cell culture medium.
Preferably, the sugars or sugar derivatives are selected from N-acetyl mannosamine and derivatives thereof, e.g. 2-desoxy-2-N-propanoylamino-D-mannose (ManNProp), 2-desoxy-2- N-Cyclopropyl-acetyl- amino-D-mannose (ManNcyProp), 2-desoxy-2-N-pentanoylamino-D- mannose (ManNPent) (see also Figures 3 and 4).
Sialylation of glycocoηjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetyl mannosamine by several consecutive steps. Using N-propanoyl mannosamine, a novel precursor of sialic acid, it is possible to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycocoηjugates taking advance of the cellular sialylation machinery. One terminal monosaccharide of nearly all glycoproteins is sialic acid. The most frequent sialic acid in humans is iV-acetylneuraminic acid (Neu5 Ac). The physiological precursor of all sialic acids is iV-acetyl D-mannosamine (ManNAc). Synthetic N-acyl-modified D- mannosamines, such as iV-propanoyl D-mannosamine (ManNProp), can be taken up by cells and efficiently metabolized to the respective iV-acyl-modified sialic acid in vitro and in vivo which can then be introduced into cellular glycoconjugates (see Figure 3).
According to the present invention varying the cell culture conditions furthermore comprises the variation of one or more of the following parameters:
(a) parameters with respect to the culture medium
- temperature different temperatures or temperature shifts
- pH value
- osmolality
- DO concentration
- inokulum cell density
(b) cultivation mode
- batch process
- fed batch process
- perfusion process
The following cell culture conditions are preferably varied depending on the cultivation mode
- batch process or fed batch process: - stirrer speed
- temperature,
- pH value
- DO concentration,
- fed batch process: - feed composition
- perfusion process: - medium flow rate,
- rotation speed,
- rotation cycle (speed and holding times).
For more detail, please see Materials and Methods in the examples. It is preferred that the bioreactor system used in the perfusion process is a liquid / gas phase exposure bioreactor family for cell cultivation as disclosed in WO2005/121311A1 and WO 2006/120202A1 (ProBioGen AG)5 which are herein incorporated by reference in their entirety. See also Langhammer et al., 2007 (ProBioGen) which is enclosed herewith by reference.
In a preferred embodiment a cell culture device and method for growing cells and for cell cultivation in high density is used, whereby the cells for cultivation are located in hollow fibre membranes and are alternately supported in a liquid nutrient and a gas phase thereabove. The device is a liquid/gas phase exposure bioreactor with a supply chamber, in which hollow fibre membranes with an inner diameter of no more than 5 mm are located and the inner volumes of which form culture chambers. After introduction of the cells into the culture chambers approximately half of the supply chamber is filled with nutrient medium and the other half with a gas mixture. After switching on the medium and gas perfusion, a cyclic exposure of the hollow fibre membranes and the cells therein to the gas or the liquid phase begins. For more details see WO2005/12131 IAl (ProBioGen).
In further preferred embodiment a device for growing and cultivating cells in membrane- based bioreactors is used which have a design that represents a scaling down of corresponding production bioreactors and which can be individually regulated with regard to temperature, pH, gas supply, mixing regime and nutrient supply. Such a device is characterised in that preferably at least six membrane-based bioreactors can be simultaneously operated in a device having a single energy supply and a centralised control and regulation unit. For more details see WO 2006/120202A1 (ProBioGen).
Furthermore, the person of skill in the art can determine further variations of the cell culture conditions when applying the teaching of the present invention.
Mammalian host cells
It is preferred that the mammalian host cell that is used in the methods of the present invention is a potent expression cell line, which has high synthetic capacity, such as high expression yields, and an extended cell viability. It is preferred that the mammalian host cell that is used in the methods of the present invention is a human neuronal cell, preferably NC5T11 and relatives thereof.
NC5T11 and relatives thereof are disclosed in WO 2007/05416 Al (ProBioGen AG), which is herein incorporated by reference in its entirety.
Preferably, a mammalian host cell suitable for the methods of the present invention, preferably NC5T11 and relatives, is a human cell, a rodent cell including mouse, rat, hamster cell, etc., more preferably is a human brain derived cell including human foetal brain cells such as foetal neurons and foetal glia cells.
Preferred mammalian host cells suitable used for the methods of the present invention, preferably NC5T11 and relatives, thereof are high expression cell lines having stably integrated into their genome a gene encoding a specific heterologous regulator protein or a functional variant thereof and stably expressing said regulator protein or the functional variant thereof.
The heterologous regulator protein preferably modulates transcription and/or cell growth, and enhances the productivity of the cell in the production of a protein differing from said regulator protein or the functional variant thereof, such as AlAT.
The functional variant of the heterologous regulator protein is preferably a fusion protein, preferably said fusion protein comprising at least one first domain comprising an adenovirus PIX regulator protein and at least one second domain that modulates or expands the activity or the subcellular distribution of the adenovirus pIX; and/or said at least one second domain comprises a protein or peptide acting as a transcription modulator, preferably said transcription modulator is a transcription factor including the retinoic acid receptor alpha, is a marker protein, preferably said marker protein is a fluorescence marker including GFP, DsRed and its variants or is an enzyme including LacZ, or is a transit peptide including a NLS.
More preferably said heterologous regulator protein (i) is the adenovirus PIX, or (ii) is a fusion protein between PIX and retinoic acid receptor alpha, or (iii) is a fusion protein between PIX5 and GFP and containing an NLS sequence, or (iv) is a fusion protein between PIX and GFP or between PIX and an isolated NLS sequence.
Furthermore, mammalian host cells suitable used for the methods of the present invention, preferably NC5T11 and relatives, carries further immortalizing (viral) genes including an El protein of an adenovirus, preferably of mastadenovirus group C type 5, most preferably the cell carries the adenovirus ElA and/or ElB gene; and/or (iv) further carries functional sequences such as selection marker sequences, splice donor/acceptor sites and/or recombinase recognition sequences allowing integration of a target nucleic acid sequence to be expressed in the cell.
Modified glycosylated human alpha- 1 antitrypsin (AlAT) aud its uses
The problem is furthermore solved by the present invention by providing glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above.
Preferably, the glycosylation status or pattern of the human alpha- 1 antitrypsin (AlAT) obtained is modulated compared to wild type human AlAT.
A "human glycosylation status or pattern" of AlAT according to the present invention means a glycosylation at the three original glycosylation sites and a glycosylation of a complex type, as e.g. described in Kolarich et al., 2006, which is incorporated herein by reference. The three original iV-glycosylation sites of AlAT contain diantennary iV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
A "modulated glycosylation status or pattern" of Al AT according to the present invention means: a) a higher and/or different degree of glycosylation,
(due to inserted and/or deleted glycosylation sites, as described in detail herein), b) an untypical high degree of a homogenous glycosylation, (e.g. lesser structure diversification or microheterogeneity of glycans) c) new sialic acids as terminal sugars (due to addition of sugars to the cell culture medium e.g. Neu5Prop instead of Neu5Ac, as described herein).
A "modulated glycosylation status or pattern" of AlAT is obtained by the methods of modulating the glycosylation status of AlAT according to the present invention, as described herein.
The human alpha- 1 antitrypsin (AlAT) obtained by the methods of this invention and which is characterized by a modulated glycosylation status or pattern is advantageous for use in treating AlAT deficiency e.g. due to an increased effective serum half life and reduced biological side effects.
The problem is furthermore solved by the present invention by using the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above, for the treatment of diseases, preferably human therapy.
The disease to be treated is selected from the group of AlAT deficiency, deficiency emphysema, neonatal cholestasis, chronic hepatic cirrhosis and cystic fibrosis. In a preferred embodiment the disease is AlAT deficiency. The person of skill in the art will be able to determine further uses of AlAT for treating and/or preventing diseases and for human therapy.
The problem is furthermore solved by the present invention by providing a method of treatment of a disease, wherein the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above is applied to a patient in need thereof.
The disease to be treated is selected from the group of AlAT deficiency, deficiency emphysema, neonatal cholestasis, chronic hepatic cirrhosis and cystic fibrosis. In a preferred embodiment the disease is AlAT deficiency. The person of skill in the art will be able to determine further treatment and/or prevention methods of diseases and for human therapy.
The problem is furthermore solved by the present invention by using the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above, for diagnosis, preferably during the above described treatment of diseases, especially in human therapy. AlAT is preferably used as diagnosis standard in a diagnosis assay for measuring plasma life time, clearance, concentration and other parameters of AlAT, preferably recombinant AlAT.
The problem is furthermore solved by the present invention by providing a diagnosis method, wherein the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention as defined above is used as a diagnosis standard for in vitro and or in vivo measuring the plasma life time, clearance, concentration and other parameters of AlAT, preferably recombinant AlAT.
The problem is furthermore solved by the present invention by providing a kit, preferably a diagnosis kit comprising the glycosylated human alpha- 1 antitrypsin (AlAT) obtained by a method according to the present invention.
In summary, advantages of the present invention are
Production of recombinant AlAT with defined oligosaccharide side chains, Production of new (modulated) glycosylated forms of AlAT with improved characteristics for treating AlAT deficiency disorder,
Expression in a human cell line (human neuronal cell line) resulting in human glycosylation of Al AT.
The following figures and examples illustrate the present invention without, however, limiting the same thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Structure of the AlAT /trypsin complex.
A ribbon diagram of the structure of alpha- 1 antitrypsin in complex with trypsin is shown, wherein AlAT is cleaved and wherein the three original glycosylation sites N46, N83 and N247 of AlAT are shown as well as loops A, B, C, D and E, which exemplify preferred sites for inserting additional glycosylation sites according to the present invention. Figure 2A and 2B. Sequence of AlAT.
The DNA sequence (SEQ ID NO. 1) and the amino acid sequence of AlAT (SEQ ID NO. 2) are shown, including the signal peptide, numbering starts at Glul (El). The loop regions are underlined. Loop C is made of 3 parts, i.e. loop Cl, loop C2 and loop C3. N* = original N- glycosylation sites (N46, N83, N247). Furthermore, mutations and the respective eight primer sequences (see also SEQ ID NOs. 3-10) are shown.
Figure 3. Schematic representation of the incorporation of unnatural sialic acids into glycoproteins involving ManNProp (2-desoxy-2-N-propanoylamino-D-mannose). Note that sialic acids are activated in the nucleus to the respective CMP-activated sialic acid. Activated sialic acids are then transported across the cytosol into the golgi compartment, where specific sialiyltransferases are located.
Figure 4. Derivates ofN-acetyl mannosamine.
The formulas of five N-acyl derivatives of D-mannosamine are shown.
Figure 5. Batch process to produce AlAT by expression in human neuronal cells. Cell count and viability in the AlAT production process with NC5T11 (also called AGEl. hn) in cellfern(pro are shown. The cells grow well with a stable viability over a period of more than 9 days.
Figure 6. Perfusion process to produce AlAT by expression in human neuronal cells. Glucose consumption and protein production per bioreactor module are shown.
EXAMPLES
Materials and Methods
1. Cell culture conditions/parameters
- pH- value from 6.5 to 7.5,
- dissolved oxygen (DO) from 10% to 200% air saturation,
- temperature from 33°C to 38°C.
2. Mode of cell culture (cultivation mode) There are two modes for culturing the mammalian host cells:
a) Batch process or fed batch process in stirred tank: cells, suspended in culture medium are given into a stirred tank and cultivated at a defined temperature, pH and DO. In batch process the cells grow and produce until the nutrients are consumed, in fed batch process some nutrients are fed to extend the vital phase of the cells. After achieving the stop criteria (defined vitality) the cell- and protein-containing medium is harvested completely.
b) Perfusion process in a membrane based bioreactor: cells are grown inside the membrane chambers of the bioreactor-module. The space surrounding the membranes (extracapillary space) is perfused continuously with medium and gas. The supply of the cells inside the membrane chambers is ensured by mass diffusion through the membrane. The AlAT can pass the membrane from inside to the extracapillary space whereas the cells are retained by the membrane. By this way the AlAT is continuously harvested with the medium perfusion flow and can be stored coolly fast.
In case a) a filtration or centrifugation is necessary to evacuate the cells from the protein solution. After this a product purification process with capture- intermediate- and polishing step follows to analyze product quality, glycosylation pattern and product yield.
It is preferred that the bioreactor system used in the perfusion process (b) is a liquid / gas phase exposure bioreactor family for cell cultivation as disclosed in WO2005/121311A1 and WO 2006/120202A1 (ProBioGen AG), which are herein incorporated by reference in their entirety.
3. Variation of cell culture conditions
- different temperatures or temperature shifts,
- different pH values and DO concentrations,
- inokulum cell density
- batch process or fed batch process (a): - stirrer speed
- temperature, - pH - DO concentration, fed batch process (a): - feed composition perfusion process (b): - medium flow rate,
- rotation speed,
- rotation cycle (speed and holding times).
Example 1 Batch process production of AlAT
NC5T11 (also called AGEl.hn) cells were cultivated in batch form in cellferm(pro) (DasGip AG, Julich, Germany). Start volume was 450ml. Cell count, viability and AlAT- concentration were measured every second day. In Figure 5 cell count and viability are shown. The cells grow well with a stable viability.
Example 2 Perfusion process production of AlAT
NC5T11 (also called AGEl.hn) cells were cultivated in a process development device of a perfusion-bioreactor for 31 days. Start cell count was 1.5 x 10 vital cells absolute per module. The flow rate was 25ml/d. Glucose consumption and protein production per day and module were measured/ calculated (see Figure 6).
The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material of realizing the invention in diverse forms thereof.
REFERENCES
Archibald AL, McClenaghan M, Hornsey V, Simons JP, Clark AJ. High-level expression of biologically active human alpha 1 -antitrypsin in the milk of transgenic mice. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5178-82. Carrell RW, Jeppsson JO, Laurell CB, Brennan SO, Owen MC, Vaughan L, Boswell DR. Structure and variation of human alpha 1 -antitrypsin. Nature. 1982 JuI 22;298(5872):329-34.
Chang GD, Chen CJ, Lin CY, Chen HC, Chen H. Improvement of glycosylation in insect cells with mammalian glycosyltransferases. JBiotechnol 2003 Apr 10; 102(1): 61-71.
Ciliberto G, Dente L, Cortese R. Cell-specific expression of a transfected human alpha 1- antitrypsin gene. Cell. 1985 Jun;41(2):531-40.
Elliott PR, Pei XY, Dafforn TR, Lomas DA. Topography of a 2.0 A structure of alphal- antitrypsin reveals targets for rational drug design to prevent conformational disease. Protein ScL 2000 Jul;9(7): 1274-81.
Higai K, Aoki Y, Azuma Y, Matsumoto K. Glycosylation of site-specific glycans of alphal- acid glycoprotein and alterations in acute and chronic inflammation. Biochim Biophys Acta. 2005 Aug 30;1725(l):128-35.
Kolarich D, Weber A, Turecek PL, Schwarz HP, Altmann F. Comprehensive glyco-proteomic analysis of human alpha 1 -antitrypsin and its charge isoforms. Proteomics 2006, 6, 3369-3380.
Langhammer S, Brecht E, Marx U. Novel bioreactors for fragile glycoproteins. Genetic Engineering News 2007, Jan 1; 27(1):34.
Paterson T, Innes J, Moore S. Approaches to maximizing stable expression of alpha 1- antitrypsin in transformed CHO cells. Appl Microbiol Biotechnol. 1994 Jan;40(5):691-8.
Song HK, Lee KN, Kwon KS, Yu MH, Suh SW. Crystal structure of an uncleaved alpha 1- antitrypsin reveals the conformation of its inhibitory reactive loop. FEBS Lett. 1995 Dec 18;377(2): 150-4.
Sudarshana MR, Plesha MA, Uratsu SL, FaIk BW, Dandekar AM, Huang TK, McDonald KA. A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues. Plant Biotechnol J. 2006 Sep;4(5):551 -9. Takeuchi M, Kobata A, Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology. 1991 Sep;l(4):337-46.
Wu Y, Foreman RC. The molecular genetics of Cc1 antitrypsin deficiency. BioEssays 1991 13(4):163-169.

Claims

Charite - Universitatsmedizin and ProBioGen AG"Methods for producing glycosylated human alpha- 1 antitrypsin (AlAT) in mammalian host cells"U30191PCTClaims
1. Method for modulating the glycosylation status of human alpha- 1 antitrypsin (AlAT), comprising culturing a mammalian host cell which expresses AlAT in cell culture under conditions that allow for protein production, wherein the glycosylation of Al AT is modulated by a) modifying the AlAT gene sequence and/or b) modifying the glycosylation machinery of the mammalian host cell and/or c) varying the cell culture conditions.
2. Method according to claim 1, wherein modifying the AlAT gene sequence comprises the insertion and/or deletion of one ore more glycosylation sites into the AlAT gene sequence.
3. Method according to claim 2, wherein the one ore more glycosylation site is a N- glycosylation site.
4. Method according to claim 2 or 3, wherein the glycosylation sites are inserted at one or more positions that are different to the original glycosylation sites within the AlAT gene sequence.
5. Method according to claim 4, wherein the one or more positions that are different to the original glycosylation sites within the AlAT gene sequence are selected from positions in the loop regions, preferably loop A, loop B, loop C, loop D and loop E.
6. Method according to claim 2 or 3, wherein the glycosylation sites are inserted and/or deleted at one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence.
7. Method according to claim 6, wherein the one or more positions within at least one of the original glycosylation domains within the AlAT gene sequence are selected from the N46 domain, N83 domain and N247 domain.
8. Method according to any one of claims 2 to 7, wherein one or more original glycosylation sites within the AlAT gene sequence are deleted.
9. Method according to claim 8, wherein the original glycosylation sites are selected from N46, N83 and N247.
10. Method according to any one of claims 2 to 9, wherein the one or more glycosylation site that is inserted is selected from the group of N48, N81, N90, N108, N123, N201, N249 and N323.
11. Method according to claim 10, wherein the one or more glycosylation site that is inserted is selected from the group of N48/T50, N81/T83, N90/T92, N108/T110, N123/T125, N201, N249/T251 and N323/T325.
12. Method according to claim 1, wherein modifying the AlAT gene sequence comprises the insertion of a glycosylation tag.
13. Method according to claim 12, wherein the glycosylation tag is selected from the group of one or more glycosylation sites of another glycoprotein, one or more glycosylation domains of another glycoprotein, one or more synthetic glycosylation domains or one or more glycosylation sites of a synthetic glycosylation domain.
14. Method according to claim 13, wherein the other glycoprotein is a highly glycosylated glycoprotein, preferably a glycoprotein with highly glycosylated glycosylation domain(s), e.g. alpha 1 -acid glycoprotein (AGP) or erythropoietin (EPO).
15. Method according to any of the preceding claims, wherein modifying the glycosylation machinery of the mammalian host cell comprises one or more of the following: a) knock out of specific glycosyltransferases, such as fucosyltransferases, b) loss-of function mutation of the epimerase/ManNAc kinase for producing a sialuria mutant, c) simultaneous co-transfection of one or several glycosidases, and/or d) knock out of specific glycosidase genes.
16. Method according to any of the preceding claims, wherein varying the cell culture conditions comprises the addition of sugars or sugar derivatives to the cell culture medium.
17. Method according to claim 16, wherein the sugars or sugar derivatives are selected from N-acetyl mannosamine and derivatives thereof, e.g. 2-desoxy-2-N-propanoylamino-D- mannose (ManNProp), 2-desoxy-2-N-Cyclopropyl-acetyl- amino-D-mannose (ManNcyProp), 2-desoxy-2-N-pentanoylamino-D-mannose (ManNPent).
18. Method according to any of the preceding claims, wherein varying the cell culture conditions comprises varying parameters with respect to the culture medium and/or the cultivation mode.
19. Method according to claim 18, wherein the parameters with respect to the culture medium are selected from temperature, pH, dissolved oxygen (DO) concentration, osmolality and inokulum cell density.
20. Method according to claim 18 or 19, wherein the cultivation mode is selected from batch process, fed batch process or perfusion process.
21. Method according to claim 20, wherein the cultivation mode is a batch process or fed batch process and the variation of the cell culture conditions is selected from stirrer speed, temperature, pH value and DO concentration.
22. Method according to claim 20, wherein the cultivation mode is a fed batch process and the variation of the cell culture conditions is a variation of the feed composition.
23. Method according to claim 20, wherein the cultivation mode is a perfusion process and the variation of the cell culture conditions is selected from medium flow rate, rotation speed and rotation cycle (speed and holding times).
24. Method according to any of the preceding claims, wherein the mammalian host cell is a human neuronal cell, preferably NC5T11 and relatives thereof.
25. Method for producing glycosylated human alpha- 1 antitrypsin (AlAT) in a mammalian host cell, further comprising i) providing a mammalian host cell, ii) providing a nucleotide sequence comprising the AlAT gene sequence, iii) transfecting said mammalian host cell with the nucleotide sequence comprising the AlAT gene sequence, iv) culturing the transfected mammalian host cell, v) obtaining AlAT expressed by the cultured mammalian host cell, wherein the glycosylation of AlAT is modulated by the method according to any of the preceding claims.
26. Glycosylated human alpha-1 antitrypsin (AlAT) obtained by a method according to any of the preceding claims, wherein the glycosylation status is modulated compared to wild type human AlAT.
27. Use of the glycosylated human alpha-1 antitrypsin (AlAT) according to claim 26 for the treatment of diseases.
28. Use according to claim 27, wherein the disease is AlAT deficiency.
29. Use of the glycosylated human alpha-1 antitrypsin (AlAT) according to claim 26 for the diagnosis, especially during treatment of disease, e.g. for measuring plasma life time, clearance, concentration of Al AT, preferably recombinant AlAT.
30. Kit for diagnosis comprising glycosylated human alpha-1 antitrypsin (AlAT) according to claim 26.
EP08773472A 2007-06-15 2008-06-16 Methods for producing glycosylated human alpha-1 antitrypsin (a1at) in mammalian host cells Withdrawn EP2167539A2 (en)

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