WO2006078645A2 - Expression de polypeptides heterologues associee a une faible multiplicite d'infection de virus - Google Patents

Expression de polypeptides heterologues associee a une faible multiplicite d'infection de virus Download PDF

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WO2006078645A2
WO2006078645A2 PCT/US2006/001582 US2006001582W WO2006078645A2 WO 2006078645 A2 WO2006078645 A2 WO 2006078645A2 US 2006001582 W US2006001582 W US 2006001582W WO 2006078645 A2 WO2006078645 A2 WO 2006078645A2
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moi
cell
virus
polypeptide
desired polypeptide
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WO2006078645A3 (fr
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Anggie Becorest
Paul M. Cino
David Hakes
Melissa Kuhar
Li Liu
Grace Mendoza
Kathleen S. Autote
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Neose Technologies, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • glycopeptides including glycosylated polypeptides, referred to herein as "glycopeptides.”
  • glycopeptides glycosylated polypeptides
  • Many expression systems have been developed to produce polypeptides and glycopeptides in eukaryotic or prokaryotic systems. Baculovirus expression system is one that is particularly useful for the production of polypeptides and glycopeptides.
  • a baculovirus expression system requires the infection of insect cells, such as Sf9 cells, with a virus containing one or more cloned nucleic acids of interest.
  • a high multiplicity of infection (MOI) is needed in order to obtain meaningful and useful levels of expression of a desired polypeptide.
  • MOI levels require a substantial input of time and resources on the front end of a virus expression system.
  • high MOI levels become exponentially problematic as the scale of the cell culture of interest increases, especially at the industrial level.
  • the present invention is based on the discovery that low MOI can be used for polypeptide production in a virus expression system. Accordingly the present invention provides methods of expressing polypeptides in a virus expression system using low MOI values.
  • the present invention provides a method of expressing a desired polypeptide in a cell. The method comprises inoculating a cell with a virus, wherein the virus comprises a nucleic acid sequence encoding a desired polypeptide and wherein the inoculation is conducted with a multiplicity of infection (MOI) at less than or equal to 0.00001.
  • MOI multiplicity of infection
  • Figure 1 is a graph illustrating the production of EPO in a baculovirus expression system, as a function of MOI and time.
  • Figure 2 shows RP-HPLC traces of the positive EPO control.
  • Figure 3 shows RP-HPLC traces of the positive EPO control.
  • Figure 4 shows RP-HPLC traces of EPO produced with an MOI of 0.0005.
  • Figure 5 shows RP-HPLC traces of EPO produced with an MOI of 0.0005.
  • Figure 6 shows RP-HPLC traces of EPO produced with an MOI of 0.005.
  • Figure 7 shows RP-HPLC traces of EPO produced with an MOI of 0.005.
  • the present invention is based on the discovery that low MOI can be used for polypeptide expressions in virus expression systems, especially virus expression systems using insect cells.
  • the present invention therefore provides methods of using low MOI values for expression or production of polypeptides in virus expression systems.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. [0020]
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double- stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • a first defined nucleic acid sequence is said to be "immediately adjacent to" a second defined nucleic acid sequence when, for example, the last nucleotide of the first nucleic acid sequence is chemically bonded to the first nucleotide of the second nucleic acid sequence through a phosphodiester bond.
  • a first defined nucleic acid sequence is also said to be "immediately adjacent to" a second defined nucleic acid sequence when, for example, the first nucleotide of the first nucleic acid sequence is chemically bonded to the last nucleotide of the second nucleic acid sequence through a phosphodiester bond.
  • a first defined polypeptide sequence is said to be "immediately adjacent to" a second defined polypeptide sequence when, for example, the last amino acid of the first polypeptide sequence is chemically bonded to the first amino acid of the second polypeptide sequence through a peptide bond.
  • a first defined polypeptide sequence is said to be "immediately adjacent to" a second defined polypeptide sequence when, for example, the first amino acid of the first polypeptide sequence is chemically bonded to the last amino acid of the second polypeptide sequence through a peptide bond.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • a "recombinant polynucleotide” is one which has been altered or produced by the hand of man.
  • a recombinant polynucleotide may be a polynucleotide isolated from a genome, a cDNA produced by the reverse transcription of an RNA, or an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • homologous refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
  • a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • a "constitutive promoter” is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell.
  • promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.
  • An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • exogenous refers to a source other than the composition in question.
  • a plasmid is considered to be derived from an exogenous source when the plasmid is transfected into a cell, e.g., an insect cell, wherein the cell previously did not contain the plasmid.
  • exogenous nucleic acid is meant that the nucleic acid has been introduced into a cell or an animal using technology which has been developed for the purpose of facilitating the introduction of a nucleic acid into a cell or an animal.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • polypeptide is used herein to refer to any amino acid polymer comprised of two or more amino acid residues linked via peptide bonds.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • “Mutants,” “derivatives,” and “variants” of the peptides of the invention are peptides which may be altered in one or more amino acids (or in one or more base pairs) such that the peptide (or nucleic acid) is not identical to the sequences recited herein, but the peptide (or peptide encoded by the DNA) has the same property as the wild type peptide or peptide of natural sequence.
  • a “variant” or “allelic or species variant” of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid.
  • two molecules possess a common activity and may substitute for each other they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
  • a "recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.
  • a "vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Expression vector refers to a vector comprising a recombinant nucleic acid comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant nucleic acid.
  • glycosyltransferase refers to any enzyme/protein that has the ability to transfer a donor sugar to an acceptor moiety.
  • a “glycopeptide” as the term is used herein refers to a peptide having at least one carbohydrate moiety covalently linked thereto. It will be understood that a glycopeptide may be a "therapeutic glycopeptide”.
  • glycopeptide is used interchangeably herein with the terms “glycopolypeptide” and "glycoprotein.”
  • "Expression” of a polypeptide means any detectable level of transcription and/or translation or activation thereof of a nucleic acid sequence encoding a polypeptide or any detectable level of the activity of a polypeptide.
  • An "antibiotic resistance marker” as the term is used herein refers to a sequence of nucleotides that encodes a protein which, when expressed in a living cell, confers to that cell the ability to live and grow in the presence of an antibiotic.
  • saccharide refers in general to any carbohydrate, a chemical entity with the most basic structure Of(CH 2 O) n - Saccharides vary in complexity, and may also include nucleic acid, amino acid, or virtually any other chemical moiety existing in biological systems.
  • Monosaccharide refers to a single unit of carbohydrate of a defined identity.
  • Oleaccharide refers to a molecule consisting of several units of carbohydrates of defined identity. Typically, saccharide sequences between 2-20 units may be referred to as oligosaccharides.
  • Polysaccharide refers to a molecule consisting of many units of carbohydrates of defined identity. However, any saccharide of two or more units may correctly be considered a polysaccharide.
  • a saccharide "donor” is a moiety that can provide a saccharide to a glycosyltransferase so that the glycosyltransferase may transfer the saccharide to a saccharide acceptor.
  • a GaINAc donor may be
  • a saccharide "acceptor” is a moiety that can accept a saccharide from a saccharide donor.
  • a glycosyltransferase can covalently couple a saccharide to a saccharide acceptor.
  • G-CSF may be a GaINAc acceptor, and a GaINAc moiety may be covalently coupled to a GaINAc acceptor by way of a GalNAc-transferase.
  • the present invention provides a method of expressing a desired polypeptide in a cell using a virus expression system.
  • the method provided by the present invention uses a low multiplicity of infection value, e.g., lower than the values normally used by skilled artisans in the field to inoculate cells for expression of desired polypeptides.
  • the value of multiplicity of infection or MOI represents the ratio between virus and cells to be infected by the virus, e.g., number of plague forming particles (pfu) per cell or per ml of cell culture.
  • a low MOI value is less than or equal to 0.00001 (10 '5 ) pfu/cell.
  • the low MOI value of the present invention is between 0.000001(10 “ 6 ) to 0.00001(10 “5 ). In another embodiment, the low MOI value of the present invention is between 0.0000001(10 “7 ) to 0.000001 (10 "6 ) or 0.0000001(10 “7 ) to 0.00001(10 "5 ). In yet another embodiment, the low MOI value of the present invention is between 0.00000001(10 “8 ) to 0.0000001(10 “7 ), 0.00000001 (10 "8 ) to 0.000001(10 "6 X or O.OOOOOOOICIO "8 ) to 0.00001(10 "5 ).
  • a cell culture such as an insect cell culture can be infected at a series of MOI values with a baculovirus expression vector containing a nucleic acid sequence encoding a desired polypeptide.
  • a cell culture such as an insect cell culture can be infected at a series of MOI values with a baculovirus expression vector containing a nucleic acid sequence encoding a desired polypeptide.
  • baculovirus expression vector containing a nucleic acid sequence encoding a desired polypeptide.
  • Means for assaying the level of expression of a desired polypeptide include, but are not limited to, enzyme activity assays, protein concentration assays, chromatography, spectroscopy, gel electrophoresis, mass spectrometry, nuclear magnetic resonance, electron paramagnetic resonance, elemental analysis, gas chromatography, differential scanning calorimetry, radioassays, immunoassays, and biological activity assays. Selection of specific methods for monitoring or characterizing expression of a desired polypeptide is based on various factors, including without any limitation, the nature, size, identity, stability, etc. of the desired polypeptide.
  • Methods for assaying the biological activity of a desired polypeptide are also available to skilled artisans in the field.
  • the Krystal assay (Krystal, 1983, Exp. Hematol. 11 :649-660) can be employed to determine the activity of EPO prepared according to the methods of the present invention.
  • the assay measures the effect of erythropoietin on intact mouse spleen cells.
  • Mice are treated with phenylhydrazine to stimulate production of erythropoietin-responsive red blood cell progenitor cells.
  • the spleens are removed, intact spleen cells are isolated and incubated with various amounts of wild-type erythropoietin or the erythropoietin proteins described herein. After an overnight incubation, 3 H-thymidine is added and its incorporation into cellular DNA is measured.
  • the amount of 3 H-thymidine incorporation is indicative of erythropoietin-stimulated production of red blood cells via interaction of erythropoietin with its cellular receptor.
  • concentration of the erythropoietin protein of the present invention, as well as the concentration of wild-type erythropoietin, is quantified by competitive radioimmunoassay methods well known in the ait. Specific activities are calculated as international units measured in the Krystal assay divided by micrograms as measured as immunoprecipitable protein by radioimmunoassay.
  • the low MOI value of the present invention can be used for expression of any desired polypeptide in a virus expression system, e.g., hormones, growth factors, enzymes, inhibitors, receptors including chimeric receptors, cytokines, etc.
  • desired polypeptides expressed using low MOI value of the present invention include exemplary polypeptides listed in Table 1. Table 1 Representative examples of polypeptides for low MOI expression
  • a desired polypeptide can be erythropoietin (EPO).
  • EPO erythropoietin
  • Any EPO may be expressed using the methods provided by the present invention, including, but not limited to human EPO.
  • EPO is an acidic glycoprotein of approximately 34 kDa and may occur in three natural forms: alpha, beta, and asialo. The alpha and beta forms differ slightly in carbohydrate components but have the same potency, biological activity and molecular weight.
  • the asialo form is an alpha or beta form with the terminal sialic acid removed.
  • U.S. Patent No. 6,187,564 describes a fusion protein comprising the amino acid sequence of two or more EPO peptides, U.S. Patent Nos.
  • desired polypeptides suitable for low MOI value in a virus expression system include members of the immunoglobulin family (e.g., antibodies, MHC molecules, T cell receptors, and the like), intercellular receptors (e.g., integrins, receptors for hormones or growth factors and the like) and lectins.
  • members of the immunoglobulin family e.g., antibodies, MHC molecules, T cell receptors, and the like
  • intercellular receptors e.g., integrins, receptors for hormones or growth factors and the like
  • lectins e.g., lectins.
  • Additional examples include renin, clotting factors such as factors V-XII, bombesin, thrombin, hematopoietic growth factor, colony stimulating factors, viral antigens, complement proteins, ⁇ l -antitrypsin, erythropoietin, P-selectin glycopeptide ligand-1 (PSGL-I), anti-thrombin III, interleukins, interferons, proteins A and C, fibrinogen, herceptin, leptin, glycosidases, HS-glycoprotein, serum proteins (e.g., ⁇ -acid glycoprotein, fetuin, ⁇ -fetal protein), ⁇ 2-glycoprotein, among many others.
  • This list of polypeptides is exemplary, not exclusive.
  • a desired polypeptide is a glycosyltransferase, e.g., a galactosyltransferase, fucosyltransferase, glucosyltransferases, N- acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases, sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases.
  • Suitable glycosyltransferases include those obtained from eukaryotes, as well as from prokaryotes.
  • a desired polypeptide can be a sialytransferase, e.g., sialytransferases listed in Table 2.
  • Table 2 Exemplary sialyltransferases which use the Gal ⁇ 1,4GIcNAc sequence as an acceptor substrate
  • a desired polypeptide can be a Gal beta-1,3 GaINAc alpha-2,3 sialyltransferase (ST3Gall), including porcine ST3Gall, as well as ST3GalI, ST3GalII, ST3GalIII, and ST3GalIV, ST3GalV, and ST3GalVI, ST6GalI, ST6GalII, ST6GalNAcII, ST6GalNAcIII, ST6GalNAcIV, ST8SiaI, ST8SiaII, ST8SiaIII, ST8SiaIV, Siat4, Siat5, Siat6, Siat7, Siat8, SiatlO, and Drosophila corel UDP- galactose:N-acetylgalactosamine-alpha-R beta 1,3-galactosyltransferase (corel GaIT).
  • ST3Gall Gal beta-1,3 GaINAc alpha-2,3 sialyltransfer
  • a desired polypeptide can be a GaINAc alpha-2, 6- sialyltransferase I (ST6GalNAcI).
  • ST6GalNAcI GaINAc alpha-2, 6- sialyltransferase I
  • Any ST6GalNAcI polypeptide may be expressed using the methods provided by the present invention, including, but not limited to human ST6GalNAcI, mouse ST6GalNAcI, and chicken ST6GalNAcI.
  • the glycosyltransferase ST6GalNAcI is an essential reagent for glycosylation of therapeutic glycopeptides. Additionally, ST6GalNAcI is an important reagent for research and development of therapeutically important glycopeptides and oligosaccharide therapeutics.
  • the low MOI value of the present invention can be used with any virus expression system, e.g., an expression system using viral infection or viral expression vector to introduce one or more exogenous nucleic acid sequences to a cell to be expressed.
  • the low MOI value of the present invention can be used with any expression system using a virus as an expression vector to infect either eukaryotic or prokaryotic cells or cell culture.
  • the virus expression system used by the present invention includes infection of a mammalian cell or cell culture with a suitable virus or viral expression vector.
  • mammalian cells or cell culture suitable for viral infection and polypeptide expression include cells or cell lines from humans, monkeys, insects, Drosophila, etc.
  • baculovirus such as Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as an expression vector for polypeptide expression in insect cells, e.g., Sf9, Sf21, Drosophila cells, e.g., Schneider S2, or commercially available cell lines, e.g., High FiveTM (Invitrogen Life Technologies, Carlsbad, CA).
  • a nucleic acid of the present invention encoding a desired polypeptide may be isolated from numerous sources, including animal or mammalian tissue, insects, nematodes, plants and cDNA libraries.
  • the isolated nucleic acid may be characterized using any technique well-known in the art, as described in detail elsewhere herein.
  • the isolated nucleic acid may be modified using well-known molecular biology technology available to the skilled artisan.
  • a desired polypeptide of the present invention is encoded by a nucleic acid comprising a nucleic acid sequence encoding the desired polypeptide.
  • a desired polypeptide of the invention for expression in a virus expression system with low MOI value should not be construed to be limited in any way.
  • a desired polypeptide of the invention may be any polypeptide, including a polypeptide having a sequence identical to the naturally-occurring form of the polypeptide, a wild type polypeptide, and a polypeptide having one or more amino acid mutations, insertions, deletions, or any combination thereof. Therefore, the present invention encompasses a desired polypeptide that has not yet been identified or expressed recombinantly.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
  • Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation.
  • glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
  • enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes.
  • sequences which have phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
  • the peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
  • a desired polypeptide of the invention is contained within a nucleic acid vector, e.g., a viral expression vector suitable for viral infection and polypeptide expression.
  • a nucleic acid vector e.g., a viral expression vector suitable for viral infection and polypeptide expression.
  • a viral expression vector suitable for viral infection and polypeptide expression.
  • Such vectors, and techniques for using and making the like, are well-known in the art, and will therefore not be discussed herein in detail.
  • Luckow et al. (1993, J. Virol. 67:4566- 4579
  • Patel et al. (1992, Nucleic Acids Res. 20:97-104
  • a desired polypeptide of the present invention may be encoded by a nucleic acid, such that the nucleic acid encoding a desired polypeptide is fused to one or more additional nucleic acids encoding a functional polypeptide.
  • an affinity tag coding sequence may be inserted into a nucleic acid vector adjacent to, upstream from, or downstream from a desired polypeptide coding sequence.
  • an affinity tag will typically be inserted into a multiple cloning site in frame with a desired polypeptide.
  • an affinity tag coding sequence can be used to produce a recombinant fusion protein by concomitantly expressing the affinity tag and desired polypeptide.
  • the expressed fusion protein can then be isolated, purified, or identified by means of the affinity tag.
  • Affinity tags useful in the present invention include, but are not limited to, a maltose binding protein, a histidine tag, a Factor IX tag, a glutathione-S-transferase tag, a FLAG-tag, and a starch binding domain tag.
  • Other tags are well known in the art, and the use of such tags in the present invention would be readily understood by the skilled artisan.
  • a vector comprising a desired polypeptide of the present invention may be used to express the desired polypeptide as either a non-fusion or as a fusion protein.
  • a non-fusion transfer vector may be used as described in Summers, et al., A Manual Of Methods For Baculovirus Vectors And Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987), incorporated herein by reference in its entirety.
  • Such vectors include, but are not limited to pVL941 (BamHI cloning site), pVL1393 (BamHI, Smal, Xbal, EcoRI, Notl, Xmalll, BgIII, and Pstl cloning site; Invitrogen; Carlsbad, CA), ⁇ VL1392 (BgIII, Pstl, Notl, Xmalll, EcoRI, Xbal, Smal, and BamHI cloning site; Invitrogen; Carlsbad, CA), and pBlueBacIII (BamHI, BgIII, Pstl, Ncol, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen; Carlsbad, CA).
  • a fusion transfer vector may also be used, such as but not limited to pAc700 (BamHI and Kpnl cloning site, in which the BamHI recognition site begins with the initiation codon), pAc701 and pAc701 (same as pAc700, with different reading frames), pAc360 (BamHI cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen; Carlsbad, CA), and pBlueBacHisA, B, C (three different reading frames, with BamHI, BgIII, Pstl, Ncol, and HindIII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen; Carlsbad, CA).
  • pAc700 BamHI and Kpnl cloning site, in which the BamHI recognition site begins with the initiation codon
  • any particular plasmid vector or other DNA vector is not a limiting factor in this invention and a wide plethora of vectors are well-known in the art. Further, it is well within the skill of the artisan to choose particular promoter/regulatory sequences and operably link those promoter/regulatory sequences to a DNA sequence encoding a desired polypeptide. Such technology is well known in the art and is described, for example, in Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • the invention thus includes a vector comprising an isolated nucleic acid encoding a desired polypeptide.
  • a desired nucleic acid into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • an isolated nucleic acid encoding a desired polypeptide is integrated into the genome of a host cell.
  • a cell is transiently transfected with an isolated nucleic acid encoding a desired polypeptide. In another aspect of the invention, a cell is stably transfected with an isolated nucleic acid encoding a desired polypeptide.
  • the invention also includes cells, viruses, proviruses, and the like, containing such vectors.
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art.
  • a nucleic acid encoding a desired polypeptide is introduced into a host cell using a virus expression system.
  • virus expression systems such as a baculovirus expression system, are well-known in the art, and will not be described in detail herein. Such virus expression systems are typically commercially available from numerous vendors.
  • a baculovirus, or a baculo virus/insect cell expression system can be used to express a desired polypeptide using a pAcGP67, pFastBac, pMelBac, or pIZ vector and a polyhedrin, plO, or OpIE3 actin promoter.
  • a Drosophila expression system can be used with a pMT or pAC5 vector and an MT or Ac5 promoter.
  • Example 1 Titration by Plaque Assay for Ervthropietin Producing Baculovirus
  • Sf-900 II SFM (IX) cells (aqueous) were incubated in a 27 0 C water bath for at least one hour prior to use.
  • EDTA solutions at pH 8.0 were prepared by adding 10 N NaOH dropwise and filtering the resultant solution through a sterilization filter unit before use.
  • MTT Staining Solution (1 mg/niL) was prepared using Dulbecco's phosphate buffered saline.
  • Sample dilutions were based on the expected baculovirus titer of the sample.
  • a sample with an expected titer of approximately 1 x 10 7 pfu/ml would be assayed at 10 "6 , 10 "7 and 10 "8 dilutions.
  • the inoculum was removed from each well of the 6-well plate and 2 mL of the agar/media solution was added rapidly by letting the solution run down the side of each well to which it was added. The plates were then incubated for 45 minutes at room temperature. At the end of the 45 minute incubation, paper towels moistened with approximately 2-4 mL of 5mM EDTA were wrapped around the plates. The plates were inverted, placed in a sterile bag and incubated at 27°C for 7-10 days, or until plaques were visible in the positive control wells to the naked eye. After 7-10 days, the plates were unwrapped and 5-7 drops of MTT staining solution was added to each well.
  • the viral titer was then determined as follows. For example, if the well representing the 10 "8 dilution had 9 colonies, the titer of the viral stock solution is 9 x 10 8 .
  • the units used were Plaque Forming Units/mL (PFU/mL). Therefore, for example, the viral titer is represented as 9 x 10 8 PFU/mL. If there were not any plaques present in the negative control and plaques were present in the positive control, then the assay was determined to be valid.
  • compositions and methods were designed to identify a plaque-purified baeulovirus clone for use in the production of EPO, wherein the MOI ranged from low to high values. In one aspect of the invention, the MOI ranged from IxIO "3 to IxIO "6 . MOI were determined as described elsewhere herein.
  • Plaque-purified baeulovirus P2 clones containing a nucleic acid sequence encoding human EPO were used in a 2 ml sample volume. Baeulovirus Sf 9 cells were obtained at passage #19, wherein the total cell concentration was 2 x 10 8 in a 50 ml culture volume.
  • AF 900 II serum-free medium was used pre- warmed to 27 0 C.
  • One ml of a P2 clone sample was added to 9 ml of fresh Sf 900 II culture medium and serially diluted five times to obtain viral dilutions of 10 "3 , 10 "4 and 10 '5 .
  • SF 9 cells (45 ml), obtained at passage #19 at 4.5 x 10 6 cells/ml, were aseptically transferred to a 50 ml conical tube and centrifuged for five minutes at 1000 rpm. The spent medium was removed from each tube and the cells were resuspended in 47 ml of fresh, pre- warmed Sf 900 II medium.
  • Three ml of virus was added to each flask in order to result in MOIs of 10 "4 to 10 "6 . Flasks were run in triplicate and centrifuged at 100 RPM, 27 0 C, for 120 hours.
  • Sf9 cells P13; 2.00e8 cells
  • Sf9 cells were resuspended in 50 mL of fresh Sf 900 II SFM medium in each of six 250 mL disposable Erlenmeyer flasks.
  • the cultures were infected with EPO virus stock as described elsewhere herein, at MOI values of 5, 0.5, 0.05, 0.005, 0.0005, and zero (i.e., no virus).
  • Cultures were incubated at 27 0 C with shaking at 130 rpm (1 inch orbit) and were sampled daily for 5 days.
  • EPO concentration was analyzed by Europium ELISA assay and reverse phase HPLC.
  • Mouse anti-human EPO antibody from Chemicon Cat# MAB 1072 is diluted to 2.5 ⁇ g/ml in 50 mM Carbonate pH 9.6 coating buffer. This coating antibody solution is dispensed at 100 ⁇ l per well in a 96 well plate and is incubated at 4 0 C overnight.
  • the plate is washed 3 times with TBS/ 0.2% Tween 20 buffer.
  • Blocking buffer (TBS/ 0.2% Tween 20/ 3% w/v dry milk) is added to each well at 200 ⁇ l per well and incubated overnight at 4 0 C. The plate is washed 3 times with TBS- Tween 0.2% buffer prior to use.
  • a standard curve using Aranesp and pegylated EPO is prepared by 1 :2 serial dilution from 25 ng/ml to 0.36 ng/ml in the appropriate sample matrix. A volume of 100 ⁇ l is dispensed in duplicate for each standard or each sample. The plate is then incubated over night at 4 0 C.
  • Europium labeled mouse anti-human EPO is diluted in PBS (IN) to a concentration of 0.8 ⁇ g/ml and 100 ⁇ l is dispensed in each well.
  • the plate is incubated at 100 rpm agitation, room temperature for 1 hour then washed 6 times with TBS/ 0.2%Tween 20 buffer.
  • the enhancement solution Perkin Elmer cat# CS500-100 is added to each well at 200 ⁇ l per well and the fluorescence is read with the Wallac plate reader.
  • Sf 9 cells were used in a culture volume of 50 ml in a 500 ml shake flask.
  • the target cell concentration used was 3e6 cells/ml, at a target MOI of: 5e-2 to 5e-6.
  • Baculovirus virus titers used included: human ST6GalNAcI clone, 5e5 pfu/ml; mouse SToGaINAcI clone, 3.75e7 pfu/ml; and chicken SToGaINAcI clone, 2.2e7 pfu/ml.
  • the total cells used in this experiment were 150e6 (3e6 per/ml, 50 ml total).
  • the total virus counts used for the highest MOIs included 7.5e6/5e5 for human (5e-2 x 150e6)/ 5e5, in 15 ml), 7.5e6/3.75e7 for mouse (5e-2 x 150e6)/3.75e7, in 0.2 ml), and 7.5e6/2.2e7 for chicken (5e-2 x 150e6)/2.2e7, 0.34 ml).
  • Virus was diluted as follows: 0.3 ml human virus + 9.7 ml Sf 900II SFM for MOI 5e-4, 1.0 ml 5e-4 + 9 ml Sf 900II SFM for MOI 5e-5, 1.0 ml 5e-5 + 9 ml Sf 900II SFM for MOI 5e-7, 1.0 ml 5e-6 + 9 ml Sf 900II SFM for MOI 5e-8, 0.4 ml mouse virus + 9.6 ml Sf 900II SFM for MOI 5e-2, and 0.68 ml human virus + 9.32 ml Sf 900II SFM for MOI 5e-2. Samples prepared for lower MOI were prepared in the same manner as described herein for the samples for the human virus.
  • Sf 9 cells were used in a culture volume of 50 ml in 500 ml shake flask.
  • the target cell concentration used was 3e6 cells/ml, and the target MOI used was 5e-5 to 5e-8.
  • Baculovirus titer used for the human ST6GalNAcI clone was 5e5 pfu/ml.
  • Total number cells used was determined to be 150e6 (3e6 per/ml x 50 ml).
  • the total number of virus particles used for the highest MOI used was 7500/5e5 ((5e-5 x 150e6)/ 5e5, m 0.015 ml).
  • virus samples were diluted as follows: 0.3 ml human virus + 9.7 ml Sf 900II SFM for MOI 5e-3, 1.0 ml 5e-3 + 9 ml Sf 900II SFM for MOI 5e-4, 1.0 ml 5e-4 + 9 ml Sf 900II SFM for MOI 5e-5, 1.0 ml 5e-5 + 9 ml Sf 900II SFM for MOI 5e-6, 1.0 ml 5e-6 + 9 ml Sf 900II SFM for MOI 5e-7, and 1.0 ml 5e-7 + 9 ml Sf 900II SFM for MOI 5e-8.
  • Example 7 Endpoint Dilution Baculovirus Titer Assay [00112] The titer of viral stocks was determined using end point dilution assay. Cells were counted and viability determined as described elsewhere herein. Cells were at least 90% viable and in log phase growth.
  • TCID 50 Reciprocal of log TCID50

Abstract

La présente invention concerne de méthodes et des compositions utilisées dans l'expression des protéines associée à de faibles valeurs de multiplicité d'infection (MOI).
PCT/US2006/001582 2005-01-19 2006-01-17 Expression de polypeptides heterologues associee a une faible multiplicite d'infection de virus WO2006078645A2 (fr)

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US7932364B2 (en) 2003-05-09 2011-04-26 Novo Nordisk A/S Compositions and methods for the preparation of human growth hormone glycosylation mutants
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US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
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US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
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