WO2011119498A1 - Production of glycoproteins in genetically modified ciliates - Google Patents

Production of glycoproteins in genetically modified ciliates Download PDF

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Publication number
WO2011119498A1
WO2011119498A1 PCT/US2011/029229 US2011029229W WO2011119498A1 WO 2011119498 A1 WO2011119498 A1 WO 2011119498A1 US 2011029229 W US2011029229 W US 2011029229W WO 2011119498 A1 WO2011119498 A1 WO 2011119498A1
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mannosidase
ciliate
seq
genetically modified
glycans
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PCT/US2011/029229
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French (fr)
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Paul Colussi
Chris Taron
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Tetragenetics, Inc.
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Publication of WO2011119498A1 publication Critical patent/WO2011119498A1/en

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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
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01113—Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase

Definitions

  • the invention relates to recombinant protein production and, in particular, methods and compositions for the production of recombinant glycoproteins in ciliates.
  • Recombinant proteins are important in a wide range of applications that extend from alternative fuel production to the treatment of human and animal diseases.
  • the manufacture of genetically engineered enzymes, therapeutic proteins, vaccines and biopolymers constitutes a multibillion dollar-per-year industry, independent of the large market for recombinant proteins produced in basic research (Pavlou and Reichert (2004), Nat. Biotechnol. 22: 1513-1519; Langer, 3 rd Annual Report and Survey of Biopharmaceutical Manufacturing, Capacity and Production, BioPlan Associates, Inc. 2005).
  • N-linked glycosylation involves the addition of a glycan moiety to an asparagine residue that resides in the following consensus sequence: Asn-X-Ser/Thr, where X can be any amino acid except proline (Kornfeld and Kornfeld (1985), Rev. Biochem. 54:631- 664).
  • N-linked glycosylation can play an essential role in many different facets of glycoprotein production and function, including correct protein folding, efficient trafficking through the secretory pathway, protein stability, and biological or therapeutic activity (Fan et al. (1997), Eur. J. Biochem.
  • composition of the N- glycans of monoclonal antibodies can play a critical role in the antibody's ability to recruit host immune system effector cells through the processes of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Shantha Raju (2008), Curr. Opin. Immun. 20:471-478; Nimmerjahn and Ravetch (2008), Nat. Rev. Immunol. 8:34-47; Jeffries and Lund (2002), Immunol. Lett. 82:57- 65; Mimura et al. (2001), J. Biol. Chem. 276:45539-45547).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc 2 core N- glycans.
  • the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc 2 core N- glycans, wherein the ciliates have significantly increased a-l,2-mannosidase activity.
  • the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc 2 core N- glycans, wherein the ciliates express a heterologous sequence encoding a protein having a- 1 ,2-mannosidase activity.
  • the protein having a-l,2-mannosidase activity comprises an endoplasmic reticulum targeting sequence.
  • the protein having a-l,2-mannosidase activity comprises a signal sequence.
  • the protein having a-l,2-mannosidase activity comprises a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 17.
  • the protein having a-l,2-mannosidase activity is selected from the group consisting of Aspergillus saitoi a-l,2-mannosidase, Trichoderma reesei a-l,2-mannosidase, Penicillium citrinum a-l,2-mannosidase, Aspergillus nidulans a- 1 ,2-mannosidase, Homo sapiens a-l,2-mannosidase I A, Homo sapiens IB a- 1,2- mannosidase, Lepidopteran insect Type I a-l,2-mannosidase, Homo sapiens a D
  • mannosidase mannosidase
  • Xanthomonas a-l,2-mannosidase
  • mouse IB a-l,2-mannosidase
  • Bacillus sp. a-l,2-mannosidase.
  • the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc 2 core N- glycans, wherein the ciliates have significantly decreased a-l,2-mannosyltransferase activity.
  • the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc 2 core N- glycans, wherein the ciliates have a micronuclear genotype comprising one or more nonfunctional Algl 1 alleles.
  • the genetic modification is selected from the group consisting of a deletion, an insertion, a substitution or an inversion in an Algl 1 allele.
  • the genetically modified ciliates are selected from the group consisting of Tetrahymena thermophila, Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi, Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis, Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes octocarinatus, Euplotes vannus, Monoeuplotes crassus, and Blepharisma japonicus.
  • the invention described herein relates to a method for producing a recombinant glycoprotein having predominantly Man 3 GlcNAc 2 core N- glycans, the method comprising: (a) transforming ciliates with an expression construct encoding the recombinant glycoprotein, (b) culturing the ciliates under conditions which promote expression of the recombinant glycoprotein, and (c) isolating the recombinant glycoprotein.
  • the recombinant glycoprotein is a human protein.
  • the recombinant glycoprotein is a therapeutic protein.
  • the recombinant glycoprotein is a cytokine.
  • the recombinant glycoprotein is a fusion protein.
  • the recombinant glycoprotein is an antibody.
  • Figure 1 is a schematic representation of the N-glycan precursor biosynthetic pathways common to many eukaryotes, including mammals and yeast, leading to the assembly of the complete glycan precursor on the dolichol lipid carrier that is transferred to a protein. Genes involved in N-glycan processing are shown by each arrow. Following transfer to the protein, the Glc 3 Man 9 GlcNAc 2 can be trimmed to remove the three glucose residues in the glycoprotin quality control pathway. A mannose can also be removed by an a-l,2-mannosidase to yield MangGlcNAc 2 prior to exit from the ER and further trafficking to the Golgi.
  • Figure 2 is a schematic representation of the maturation of yeast
  • Figure 3 is a schematic representation of the maturation of mammalian Man 8 GlcNAc 2 N-glycans in the Golgi, which produces complex glycans. Genes involved in N-glycan processing are shown above each arrow.
  • Figure 4 is a schematic representation of predicted ciliate N-glycan precursor biosynthetic pathway. Genes involved in N-glycan processing are shown above each arrow.
  • Figure 5 is a schematic representation of the precursor glycans produced in all fungi, plants and animals (Glc3Man9GlcNAc 2 ) and those produced in ciliates
  • FIG. 6 is a schematic representation of the design of an expression cassette used for the expression of a-l,2-mannosidases in a ciliate, Tetrahymena.
  • DNA targeting sequences (5' BTUl and 3' BTUl) were used to direct integration of the cassette at the Tetrahymena BTUl locus.
  • a cycloheximide resistance cassette (Cyc r ) in relation to the Catalase promoter (pr) and the a-l,2-mannosidase gene.
  • the 3' BTU1 targeting DNA provides a terminator for the expression of the a-l,2-mannosidase gene.
  • FIG. 7 shows results of experiments in which the a-l,2-mannosidase from Entamoeba histolytica was expressed in a ciliate.
  • A. E. histolytica a-l,2-mannosidase was expressed in Tetrahymena. Tetrahymena cell lysates were prepared from a strain containing an integrated a-l,2-mannosidase expression cassette. Anti-HA western blot analysis indicates that transgenic strains express the recombinant protein. No signal is detected in wild-type cells (WT).
  • WT wild-type cells
  • B Recombinant E. histolytica a-l,2-mannosidase is localized in the Tetrahymena endoplasmic reticulum (ER).
  • Top left panel shows Tetrahymena nuclei only (DAPI staining).
  • Top right panel shows anti-HA detection of mannosidase.
  • Bottom left panel shows a merged image of the top two panels. Staining patterns are indicative of an ER localized protein.
  • Figure 8 shows results of experiments in which a-l,2-mannosidase significantly increases the relative amount of Man3GlcNAc 2 glycans attached to
  • glycoproteins when expressed in Tetrahymena Mature glycoproteins from Tetrahymena wild-type strains and transgenic strains expressing a-l,2-mannosidase were purified from spent culture medium. N-linked glycans were released from purified proteins by treatment with PNGaseF and analyzed by high-pressure anion exchange chromatography. Peaks in chromatograms are labeled in red and corresponding structures are shown in figure inserts. The relative amounts of each glycan are shown circled in red. In wild-type cells the total amount of Man3GlcNAc 2 glycan isolated represents approximately 44% total glycan. In transgenic strains Man3GlcNAc 2 glycan represents approximately 82% total glycan.
  • Figure 9 shows results of experiments in which attenuation of Algl 1 function in Tetrahymena results in an increase in relative Man3GlcNAc 2 glycan associated with glycoprotein.
  • Mature glycoproteins from a Tetrahymena strain that contains disrupted alleles of the Algl 1 gene were purified from spent culture medium.
  • N-linked glycans were released from purified proteins by treatment with PNGaseF and analyzed by high-pressure anion exchange chromatography. Peaks are labeled in red and corresponding structures are shown in the figure insert. The relative amounts of each glycan are shown circled in red.
  • glycosylation of a recombinant protein can vary depending upon the expression system used, and the particular conditions used to produce the given protein. Controlling the type and extent of protein glycosylation can be important for the expression of functional recombinant proteins. Production of polypeptides in non-human sources can result in non-human glycosylation of the protein and, thus, immunogenic responses if the protein is administered to a human subject.
  • compositions immunogenic (Patel et al. (1992), Biochem. J. 285:839-845).
  • differential glycoforms can also result in pharmacokinetic and pharmacodynamic problems including, but no limited to, issues relating to receptor-interaction, tissue-specific targeting, protease resistance, serum half life, and complement binding (Graddis et al. (2002), Curr. Pharm. Biotechnol. 3:285-297; Jefferis and Lund (1997), Antibody Eng. Chem. Immunol. 65: 111-128; Wright and Morrison (1997), Trends Biotechnol. 15:26-32).
  • the methods and compositions described herein relate to methods for generating genetically modified ciliates ⁇ e.g., Tetrahymena thermophila) that are capable of substantially uniform glycosylation with glycans comprising distinct carbohydrate structures.
  • the ciliates have a genetically modified glycosylation pathway such that recombinant glycoproteins expressed in these cells display predominantly or substantially uniformly a Man 3 GlcNAc 2 core N-glycan structure.
  • these genetically modified ciliates can be obtained using a combination of genetic engineering and/or selection of strains which have reduced a- 1,2- mannosyltransferase and/or increased a-l ,2-mannosidase activity than a ciliate that has not been genetically modified.
  • the methods described herein provide a method for producing one or more glycoproteins containing predominantly Man 3 GlcNAc 2 core N- glycans. In another aspect, the methods described herein provide a method for producing one or more glycoproteins containing substantially uniform Man 3 GlcNAc 2 core N-glycans. In general, the methods described herein relate to the use of genetically modified ciliates as an expression system for producing glycoproteins, wherein the genetically modified ciliates comprise one or more modifications of the glycosylation pathway such that all, or a predominant molar ratio, of the glycoproteins produced by the ciliate will have
  • compositions described herein are based in part on the finding that increased a-l ,2-mannosidase activity and/or reduced a-l ,2-mannosyltransferase activity in a ciliate will result in all, or a predominant molar ratio, of the glycoproteins produced by the ciliate having Man 3 GlcNAc 2 core N-glycans.
  • the genetically modified ciliates described herein can comprise one or more deletions or disruptions of one or more copies of an Algl 1 gene locus.
  • deletion or disruption of the Algl 1 gene locus results in the generation of a genetically modified ciliate capable of producing a recombinant polypeptide (e.g. , a therapeutic polypeptide or an antibody) in which Man 3 GlcNAc 2 is the predominant core N- glycan.
  • a ciliate comprising one or more deletions or disruptions of an Algl 1 gene can be further genetically modified so as to have greater a-l ,2-mannosidase activity than a ciliate that has not been genetically modified.
  • the genetically modified ciliates described herein express a transgene encoding a-l ,2-mannosidase, wherein expression of the a- 1 ,2- mannosidase causes the ciliate to produce glycoproteins having substantially uniform
  • the genetically modified ciliates described herein express a transgene encoding a-l ,2-mannosidase and wherein expression of the a-l ,2-mannosidase causes the ciliate to produce glycoproteins having predominantly Man 3 GlcNAc 2 core N-glycans.
  • the genetically modified ciliates described herein comprise at least one modified Algl 1 gene locus which causes reduced a- 1,2- mannosyltransferase activity in the cell, thereby causing the ciliate to produce glycoproteins having predominantly Man3GlcNAc 2 core N-glycans.
  • the genetically modified ciliates described herein have at least one modified Algl 1 gene locus causing reduced a-l,2-mannosyltransferase activity, or complete loss of a- 1,2- mannosyltransferase activity in the cell, thereby causing the ciliates to produce glycoproteins having substantially uniform Man 3 GlcNAc 2 core N-glycans.
  • the invention provides for glycoproteins produced by the genetically modified ciliates described herein.
  • the glycoprotein is a therapeutic polypeptide.
  • the glycoprotein is an antibody.
  • GlcN - glucosamine
  • GlcNAc N-acetylglucosaminyl
  • GalNAc N-acetylgalactosamine
  • ManN mannosamine
  • ManNAc N-acetylmannosamine
  • Sia sialic acid
  • Neu5AC N-acetylneuraminic acid (NANA)
  • Neu5Gc N-glycolylneuraminic acid
  • GlcA glucuronic acid
  • the term "ciliate” means a eukaryote belonging to the kingdom Chromalveolata, the superphylum Alveolata, and the phylum Ciliophora. Ciliates are complex protozoa characterized by the presence of cilia on their cell surfaces and dimorphic nuclei consisting of a macronucleus and one or more micronuclei.
  • Tetrahymena spp refers to ciliate protozoa in the family of Tetrahymenidae.
  • Exemplary Tetrahymena spp. include but are not limited to,
  • thermophila and T. pyriformis.
  • N-glycan refers to an N-linked oligosaccharide, including, for example, one that is or was attached by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a polypeptide.
  • N-glycans can differ with respect to the number of branches (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branches (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branches (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branch (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branch (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branch (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, Gal, Fuc, Sia) that are added to the branch (antenna
  • N-glycans can be classified according to their branched constituents ⁇ e.g., high mannose, complex or hybrid). For example, a "high mannose" type N-glycan can have five or more mannose residues.
  • a "complex" type N-glycan can have at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a Man 3 GlcNAc 2 core.
  • Complex N-glycans can also have Gal or GalNAc residues that can be further modified with Sia or derivatives ⁇ e.g., NeuAc).
  • the various N- glycans are also referred to as "glycoforms.”
  • core N-glycan means (1) any glycan structure, comprising a multiplicity of saccharide residues with stereospecific linkages, that is transferred from a dolichol pyrophosphate (Dol-PP) glycan precursor to a nascent
  • the structure of a processed core N-glycan includes any oligosaccharide residues (with the same stereospecific linkages) which are found in the initial core N-glycan, but excludes any oligosaccharide residues which were added or removed during glycosyl processing subsequent to transfer of the initial core N-glycan from the Dol-PP glycan precursor.
  • the Glc 3 Man9GlcNAc 2 glycan is the initial core N-glycan for yeast and mammals.
  • the processed core N-glycans of yeast and mammals include the Man 8 GlcNAc 2 and Man 3 GlcNAc 2 glycan substructures shown after steps Y4 and M10, respectively.
  • the Glc 3 Man5GlcNAc 2 glycan is the initial core M- glycan in ciliates.
  • the processed core N-glycans of ciliates include the Man 5 GlcNAc 2 , Man 4 GlcNAc 2 and Man 3 GlcNAc 2 glycan substructures identified
  • operably joined refers to a covalent and functional linkage of one or more genetic regulatory elements and one or more genetic coding regions which can cause the coding region(s) to be transcribed into mRNA by an RNA polymerase which can bind to one or more of the regulatory elements.
  • a regulatory region including regulatory elements such as a promoter and/or enhancer, is operably joined to a coding region when an RNA polymerase is capable of binding under permissive conditions to a promoter within the regulatory region and causing transcription of the coding region(s) into mRNA.
  • permissive conditions would include standard intracellular conditions for constitutive promoters, standard conditions and the absence of a repressor or the presence of an inducer for repressible/inducible promoters, and appropriate in vitro conditions, as known in the art, for in vitro transcription systems.
  • heterologous means, with respect to two or more genetic or protein sequences, that the sequences do not occur in the same physical relation to each other in nature and/or do not naturally occur within the same genome or protein.
  • a genetic construct may include a coding sequence which is operably joined to one or more regulatory sequences, or to one or more other coding sequences, and these sequences are considered heterologous to each other if they are not operably joined in nature and/or they are not found in the same relation in a genome in nature.
  • a protein may include a first polypeptide sequence which is joined by a standard polypeptide bond to a second polypeptide sequence, and these sequences are considered heterologous to each other if they are not found in the same relation in any protein or proteome in nature.
  • the term “recombinant” means, with respect to a protein, that the protein comprises two or more heterologous amino acid sequences, which are encoded by a DNA molecule (e.g. , a plasmid or a chromosome) comprising two or more heterologous nucleic acid sequences, or that the protein is heterologous to the cell in which it is produced.
  • the term “recombinant” means that the cell includes a DNA molecule (e.g., a plasmid or a chromosome) comprising at least one heterologous nucleic acid sequence.
  • promoter means a nucleotide sequence which is capable of binding an R A polymerase and initiating transcription of a downstream or 3' coding sequence.
  • selectable marker means any genetic sequence which, when expressed, has a biochemical or phenotypic effect which is dominant and selectable by the presence or absence of a selection agent or by physial means (e.g., phenotypic sorting).
  • Selectable marker genes that confer resistance or tolerance to a normally toxic selection agent cause only successfully transfected cells to survive in the presence of the selection agent and are referred to as positive selection markers.
  • Selectable marker genes that confer sensitivity or susceptibility to a normally non-toxic selection agent cause only successfully transfected cells to die in the presence of the selection agent are referred to as negative selection markers.
  • Phenotypic selectable marker genes permit selection based upon morphological or biochemical traits rather than cell death or survival. In some cases, the phenotypic marker is detectable only in the presence of an additional selection agent (e.g., a chromogenic substrate for an enzyme).
  • an additional selection agent e.g., a chromogenic substrate for an enzyme
  • vector means any genetic construct, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of transferring nucleic acids into or between cells.
  • Vectors may be capable of one or more of replication, expression, and insertion or integration, but need not possess each of these capabilities.
  • the term includes cloning, expression, homologous recombination, and knock-out vectors.
  • expression vector means a vector comprising regulatory elements operably joined to one or more coding regions to be expressed in a host cell transformed with the expression vector.
  • transformation means to introduce into a cell an exogenous nucleic acid or nucleic acid analog that encodes a polypeptide sequence which is expressed in that cell (with or without integration into the genome of the cell), and/or that is integrated into the genome of that cell so as to affect the expression of a genetic locus already present within the genome.
  • transformation is used to embrace all of the various methods of introducing such nucleic acids or nucleic acid analogs, including, but not limited to the methods referred to in the art as transformation, transfection, transduction, or gene transfer, and including techniques such as microinjection, DEAE-dextran-mediated endocytosis, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, ballistic injection, viral-mediated transfection, particle bombardment and the like.
  • polypeptide means an oligopeptide, polypeptide, or protein.
  • polypeptide herein can refer to a polypeptide either with and without glycosylation.
  • glycosypeptide means a polypeptide chain having one or more glycosyl moieties attached thereto. No distinction is made herein to differentiate small glycopolypeptides from large glycopolypeptides or glycoproteins.
  • antibody means a naturally produced antibody, recombinantly produced antibody, monoclonal antibody, or polyclonal antibody, as well as an antibody fragment such as an Fab fragment, F(ab') 2 fragment, Fv fragment, or single-chain Fv fragment (scFv) that retains the relevant binding specificity of the native antibody.
  • antibody is used to include antibodies of all immunoglobulin classes, such as IgM, IgG, IgD, IgE, IgA, and their subclasses.
  • the term "predominant" indicates greater than 50%.
  • the terms “increase” and “decrease” mean, respectively, to cause an increase or decrease of at least 5%, as determined by a method and sample size that achieves statistical significance (i.e., p ⁇ 0.01).
  • the term "statistically significant” means having a probability of less than 1% under the relevant null hypothesis (i.e., p ⁇ 0.01).
  • variable can be equal to any integer value within the numerical range, including the end-points of the range.
  • variable can be equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, . . . , 0.9, 0.99, 0.999, or any other real values > 0 and ⁇ 2, if the variable is inherently continuous.
  • the present invention depends, in part, upon the discovery that the glycoslyation pathways of ciliates can be genetically modified to produce glycoproteins in which the glycans have a predominant core N-glycan structure which includes a
  • Man 3 GlcNAc 2 N-linkage In some embodiments, the mature glycoproteins comprise predominantly Man 3 GlcNAc 2 N-linked glycans. In other embodiments, a Man 3 GlcNAc 2 glycan is produced as an intermediate that is subsequently modified by additional endogenous or heterologous glycosylation enzymes, resulting in a processed glycan which retains the Man 3 GlcNAc 2 core N-glycan.
  • Recombinant expression systems suitable for the production of clinically and economically important proteins using the methods described herein include, but are not limited to, recombinant protein expression in Tetrahymena thermophila,
  • N-linked glycan biosynthesis occurs in a step-wise pathway in the endoplasmic reticulum and Golgi apparatus in a process that is well-conserved among many eukaryotes (for a review, see Chen et al. (2005), "Mammalian Glycosylation: An overview of carbohydrate biosynthesis," in Yarema, ed., Handbook of Carbohydrate Engineering, Boca Raton (FL), Dekkar CRC Press).
  • glycan precursor In yeast and mammalian cells, assembly of the glycan precursor is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) membrane on a dolichol pyrophosphate (Dol-PP) isoprenoid lipid carrier by the addition of two GlcNAc and five Man residues ( Figure 1). The Man 5 GlcNAc 2 -Dol-PP oligosaccharide is then flipped to the luminal face of the ER.
  • Dol-PP dolichol pyrophosphate
  • Figure 1 The Man 5 GlcNAc 2 -Dol-PP oligosaccharide is then flipped to the luminal face of the ER.
  • the glycans may be further modified by processing enzymes that add and/or remove sugars from the glycan structure.
  • processing enzymes that add and/or remove sugars from the glycan structure.
  • the nature and extent of these glycan remodeling reactions can differ.
  • yeast the addition in the Golgi of a single a-l,6-linked mannose to the a- 1,3 branch of the core glycan by the mannosyltransferase Ochl initiates synthesis of outer chain mannan, a structure that can decorate the glycan with well over 100 additional mannoses (Figure 2).
  • Mannose addition is achieved through the action of a number of different mannosyltransferases (e.g., Mnnl, Mnn2, Mnn5, Mnn9, MnnlO, Mnnl 1, Anpl, Vanl, Hocl) and the resulting hyper- mannosylated glycans tend to be largely heterogeneous in nature (Jigami (2008), Biosci. Biotechnol. Biochem. 72:637-648).
  • Mnnl, Mnn2, Mnn5, Mnn9, MnnlO, Mnnl 1, Anpl, Vanl, Hocl mannosylated glycans tend to be largely heterogeneous in nature
  • N-glycans are processed to generate either hybrid or complex-type glycans ( Figure 3). In both cases, the cis-Golgi mannosidase I first removes a- 1,2-linked mannoses to generate a MansGlcNAc 2 oligosaccharide.
  • a hybrid glycan is formed in the medial-Golgi by addition of a single a-l,2-linked GlcNAc to the a-l,3-linked mannose by GlcNAc transferase I.
  • the remaining two outer mannose residues (a-1,3 and a-1,6) are subsequently removed through the action of mannosidase II to generate a
  • GlcNAciMan 3 GlcNAc 2 oligosaccharide.
  • a second a- 1,2-linked GlcNAc residue is then added by GlcNAc transferase II to generate a GlcNAc 2 Man 3 GlcNAc 2 complex-type glycan.
  • This is followed by the addition of two ⁇ -1,4 galactose residues by a galactose transferase and finally by the addition of terminal sialic acids by various sialyl transferases.
  • complex glycan structures may display significant diversity, including tri and tetra-antennary structures that may or may not contain additional side-branching moieties such as a a-1,6 fucose (added in the medial Golgi by fucosyl transferase) or bisecting ⁇ -1,4 GlcNAc.
  • the typical processed core N-glycan is Man 3 GlcNAc 2 .
  • composition and uniformity of N-linked glycosylation can be important for the production of therapeutic recombinant proteins.
  • glycan uniformity can be difficult to control in mammalian heterologous protein expression systems such as CHO cells.
  • Most glycoprotein therapeutics that are currently approved for human use contain a mixture of several different N-glycan structures.
  • yeast N-glycosylation pathways Recently, systematic engineering of yeast N-glycosylation pathways (termed “glycoengineering”) has resulted in the expression of glycoproteins bearing humanlike glycosylation pathways.
  • Such methods have been used to produce a protein bearing a fully sialylated N-glycan with the structure NANA 2 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 in a yeast strain deficient in Ochl, to eliminate the outer chain mannan on yeast N-glycans (Gerngross (2004), Nat. Biotechnol. 22: 1409-1414; Li et al. (2006) Nat. Biotechnol. 24:210-215;
  • Ciliates are complex unicellular eukaryotes.
  • annotation of the fully sequenced genome and EST analysis of the ciliate Tetrahymena thermophila indicates that this organism has approximately 24,000 genes (Eisen et al. (2006), PLoS Biol. 4: 1620-1642; Coyne et al. (2008), BMC Genomics 9:562).
  • Large extended gene families of ABC transporters, GPI-anchored proteins and voltage-gated ion channels are indicative of the complexity of ciliates. Indeed the presence in T. thermophila of over 400 potassium ion channels rivals that of human cells.
  • ciliates in general, and Tetrahymena still enjoy the benefits of a microbial expression platform, including fast generation times (1.5-3h), scalability, and growth on a number of inexpensive complex and defined media.
  • Figure 4 is a schematic representation of the putative major pathway of N- glycan synthesis in ciliates, and illustrates the differences from the putative major pathway in yeast and mammalian cells depicted in Figure 1. Specifically, comparing Figures 1 and 4, although ciliates contain the enzymes necessary for steps 1 through 8 shown in both figures, they lack the enzymes necessary for steps 9 through 12 of Figure 1. Thus, like yeast and mammalian cells, the primary glycosylation pathway leads to the production of a
  • ciliates as in yeast and mammalian cells, after the glycan Dol-PP oligosaccharide is transferred to a protein by an oligosaccharyltransferase complex (OST), the three Glc residues are removed by ER resident a glucosidases I and II.
  • OST oligosaccharyltransferase complex
  • ciliates lack a classic Golgi apparatus or Golgi bodies (see, e.g., Elliott and Zieg (1968), J. Cell Biol. 36(2):391-398) and, therefore, Golgi- associated enzymes, they do not produce the elaborate complex and hybrid structures generated in the Golgi of yeast or mammalian cells ( Figures 2 and 3).
  • Additional processing can include removal of up to three mannose residues by one or more yet unidentified enzymes with a-l,2-mannosidase, a-l,3-mannosidase and/or a-l,6-mannosidase activity. Such mannose trimming may occur intracellulary in the compartment of the secretory pathway and/or in the extracellular media.
  • the putative major glycosylation pathway in ciliates described above is also consistent with bioinformatic analysis.
  • sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia reveals homologs of all the yeast and mammalian genes required to produce N-glycans as shown in steps 1 through 16 of Figure 1 with the notable absence of the genes encoding the Alg3, Alg9 and Algl2- mannosyltransferases required for steps 9 through 12.
  • the ciliates lack homologs to many other yeast and mammalian genes involved in glycosylation (Table 1).
  • N-glycan precursor synthesis enzymes N-glycan precursor synthesis enzymes
  • the OST complex in yeast contains 9 subunits (Ostlp-Ost6p, Stt3p, Swplp and Wbplp) with only 5 being essential for viability (Ostlp, Ost2p, Stt3p, Swplp and Wbplp).
  • Other non-essential subunits are typically short hydrophobic peptides and are therefore difficult to identify in the genomes of distantly related organisms using Blast homology analysis. Therefore, only the essential components of OST are shown in this table to illustrate that some related form of OST is present in ciliates.
  • processing of the glycan precursor in ciliates involves only removal of the three Glc residues followed by mannose trimming through the action of a protein having a-l,2-mannosidase activity that generates Man 4 GlcNAc 2 and Man3GlcNAc 2 glycans, and either an a- 1,3 or an a-l,6-mannosidase that generates Man 2 GlcNAc 2 glycans.
  • this processing of the ciliate glycans is occurring.
  • This activity occurs in late stage secretory vesicles following transit of glycoproteins from the ER.
  • the present invention provides genetically modified ciliates which produce glycoproteins with predominantly or substantially uniform Man 3 GlcNAc 2 core N-glycans. In some embodiments, this is achieved through the production of genetically modified ciliates that express an exogenous a-l,2-mannosidase which significantly increases the percentage of Man 3 GlcNAc 2 core N-glycans relative to Man4GlcNAc 2 and MansGlcNAc2 core N-glycans.
  • the invention can achieve different percentages of Man 3 GlcNAc 2 core N-glycans on glycoproteins produced by the ciliates.
  • the glycoproteins produced by the genetically-modified ciliates will have predominantly Man 3 GlcNAc 2 core N-glycans.
  • the percentage of total N-glycans having a Man 3 GlcNAc 2 core N-glycan structure can range from 50%- 100%, including all values in between.
  • the percentage of Man 3 GlcNAc 2 core N-glycans can be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the a-1 ,2-mannosidase, or protein having a-1 ,2-mannosidase activity used in conjunction with the methods described herein, can be from any source.
  • an expression cassette can be designed to express an a-l,2-mannosidase (GenBank Gene ID, 3408991) from the protist Entamoeba histolytica.
  • a-l,2-mannosidases suitable for use in the methods described herein include, but are not limited to: Aspergillus saitoi a- 1,2- mannosidase (Ichishima et al. (1999), Biochem. J. 339(Pt 3):589-597), Trichoderma reesei a-l,2-mannosidase (Maras et al. (2000), J. Biotechnol. 77(2-3):255-263), Penicillium citrinum a-l,2-mannosidase (Yoshida et al. (1993), Biochem. J. 290(Pt 2):349-354),
  • Aspergillus nidulans a-l,2-mannosidase (Eades and Hintz (2000), Gene 255(l):25-34), Homo sapiens a-l,2-mannosidase I A, Homo sapiens IB a- 1,2- mannosidase, Lepidopteran insect Type I a-l,2-mannosidase (Ren et al. (1995), Biochem. 34(8):2489-2495), Homo sapiens aD mannosidase (Chandrasekaran et al. (1984), Cancer Res.
  • an a-l,2-mannosidase is expressed in a ciliate and targeted to a site in the secretory pathway where at least one
  • Man 3 GlcNAc 2 N-glycan structure at least one Man 4 GlcNAc 2 N-glycan structure (a substrate of a-l,2-mannosidase), or at least one Man 5 GlcNAc 2 N-glycan structure (a substrate of a- 1,2- mannosidase) is present.
  • the a-l,2-mannosidase is targeted to the ER.
  • the a-l,2-mannosidase is targeted to cortical granules or mucocysts.
  • a polypeptide having a-l,2-mannosidase activity can be linked to an ER-targeting signal that causes the polypeptide to be targeted to and/or retained in the ER or ER-derived storage or secretory vesicle.
  • the a-l,2-mannosidase is localized to the ciliate ER through targeting sequences which are native to the mannosidase-coding sequences.
  • the ER targeting sequences are heterologous to the mannosidase-coding sequences.
  • An "ER targeting sequence” refers to a peptide sequence which directs a protein having such a peptide sequence to be transported to and/or retained in the ER or an ER-derived storage or secretory vesicle. Such ER targeting sequences are often found in proteins that reside and function in the ER, and multiple such ER targeting signals suitable for use with the methods described herein can readily be identified by one skilled in the art.
  • the ER targeting sequences can be N-terminal or C-terminal.
  • a targeting sequence that is capable of retaining a polypeptide in the ER contains a C-terminal ER-retention motif.
  • C-terminal ER-retention motifs include KDEL, HDEL, DDEL, ADEL and SDEL sequences, as well as those ER-retention sequences disclosed in Xie et al. (2007), Eukaryotic Cell 6(3):388-397 and Andres et al. (1991), J. Biol. Chem. 266(22): 14277-14282.
  • targeting sequences are well known and described in the scientific literature and public databases (see http://www.ciliate.org/genetics.shtml).
  • Such targeting sequences include, but are not limited to, the ER retention sequences from the Tetrahymena GPI transamidase complex.
  • the GPI transamidase complex is involved in the transfer of lipid-linked GPI precursor molecules to target proteins in the ER.
  • the C- terminal portion (54 amino acids) of the PigT component of the GPI transamidase complex (which includes a transmembrane domain), can be fused to a recombinant protein to maintain ER localization.
  • any Tetrahymena ER resident protein, or portion thereof, having ER retention activity can be used as a fusion partner to promote ER localization of the fusion partner.
  • an a-l,2-mannosidase from a non-ciliate species may not have an ER targeting sequence, or will not have an ER targeting sequence which functions well in a ciliate. In such cases, one skilled in the art will readily be able to modify such an a-l,2-mannosidase from a non-ciliate species.
  • ER targeting sequence by adding an appropriate ER targeting sequence to the a-l,2-mannosidase by standard protein engineering techniques.
  • the a-l,2-mannosidase contains one or more non-ER targeting signals (e.g., a signal that may direct the a-l,2-mannosidase to another subcellular compartment or organelle)
  • non-ER targeting signals e.g., a signal that may direct the a-l,2-mannosidase to another subcellular compartment or organelle
  • one skilled in the art will readily be able to modify the protein to remove any non-ER targeting sequences and to add an appropriate ER targeting sequence (e.g., as disclosed in Xie et al. (2007), Eukaryotic Cell 6(3):388-397).
  • the a-l,2-mannosidases can further comprise targeting sequences that direct localization of the a-l,2-mannosidase to one or more other subcellular compartments or organelles.
  • the a-l,2-mannosidase can be localized to a ciliate's mucocysts through fusion of the a-l,2-mannosidase to a polypeptide having a mucocyst retention or targeting signal.
  • Exemplary polypeptides having mucocyst targeting sequences suitable for use with the methods described herein include both full length granule lattice proteins (e.g., Girl), the "pre-pro" domains of granule lattice proteins, and Igr proteins.
  • Ciliates that include mucocysts useful in the invention include
  • Tetrahymena species such as Tetrahymena thermophila and Tetrahymena pyriformis. Both Tetrahymena thermophila and Tetrahymena pyriformis produce mucocysts, and both can produce a heterologous protein which will be secreted within the proteinaceous gel which results from mucocyst discharge. Paramecium has dense core granules but does not secrete a proteinaceous gel.
  • the a-l,2-mannosidase can be localized to a ciliate constitutive secretory pathway either through localization sequences native to the a- 1,2- mannosidase or through fusion of the mannosidase to a polypeptide having a constitutive secretory pathway targeting signal.
  • An exemplary polypeptide having a secretory pathway targeting signal is the signal peptide from the immobilization antigen variant B protein of Ichthyophthirius multifiliis (MKFNILIILIISLFINELRA (SEQ ID NO: 1)).
  • Other polypeptide sequences having secretory pathway targeting activity can be identified by those skilled in the art using standard algorithms (e.g. , SignalP) suitable for detecting the presence and location of signal peptides in amino acid sequences.
  • Exemplary Tetrahymena polypeptides having secretory pathway targeting activity include, but are not limited to those polypeptides listed in Table 2.
  • the a-l,2-mannosidase does not contain any native or heterologous targeting or localization sequences.
  • the a-1 ,2-mannosidase for use in the present invention can be further modified by, for example, insertion of an epitope tag to which antibodies are available ⁇ e.g. , Myc, HA, FLAG and His6 tags) by methods well-known in the art.
  • An epitope-tagged a- 1,2- mannosidase can be conveniently purified, or monitored for both expression and intracellular localization.
  • the a-l,2-mannosidase is expressed from an expression cassette suitable for expressing the a-l,2-mannosidase in the ciliate.
  • expression cassettes can comprise regulatory elements, such as a promoter and/or a terminator, as well as optional sequences for positive or negative selection, such as sequences conferring resistance or susceptibility.
  • Such expression cassettes can be maintained in either the ciliate macronucleus or in the micronucleus.
  • the expression cassette encoding a protein having a-l,2-mannosidase activity can further comprise a transgene for the expression of a recombinant polypeptide of interest (e.g., a therapeutic polypeptide).
  • a transgene for the expression of a recombinant polypeptide of interest e.g., a therapeutic polypeptide.
  • Polypeptides encoded by transgenes suitable for expression by the genetically modified ciliates described herein are not limited to any particular protein and can include any polypeptide which it is desirable to produce with predominantly or substantially uniform Man 3 GlcNAc 2 core N-glycans.
  • Any method known in the art can be used to select for the genetically modified ciliates described herein. Suitable methods include, but are not limited to, selection for resistance to an antibiotic. In certain embodiments, phenotypic assortment can also be used to select for one or more genetically modified ciliates suitable for use with the methods described herein.
  • the genetically modified ciliates having significantly increased a- 1,2- mannosidase activity can also have significantly reduced a-l,2-mannosyltransferase activity, as described below.
  • Algl 1 is an a-l,2-mannosyltransferase that sequentially adds two a-l,2-linked mannose residues to the Man 3 GlcNAc 2 Dol-PP glycan precursor on the cytoplasmic face of the ER to produce a MansGlcNAc 2 Dol-PP glycan precursor, which is then translocated to the luminal face of the ER.
  • ciliates as shown in Figure 4, no further mannose residues are added before the enzymes Alg6, Alg8 and AlglO sequentially add three glucose residues to the terminal a-l,2-linked mannose residue prior to transfer of the initial core N-glycan to the nascent glycoprotein.
  • Alg6 and AlglO sequentially add three glucose residues to the terminal a-l,2-linked mannose residue prior to transfer of the initial core N-glycan to the nascent glycoprotein.
  • the invention provides a glycoprotein expression system wherein genetically engineered ciliates, in which at least one Algl 1 gene locus has been deleted, disrupted or modified to have significantly reduced expression, are used to produce the glycoproteins containing predominantly Man 3 GlcNAc 2 core N-glycans.
  • the invention provides a glycoprotein expression system wherein genetically engineered ciliates having substantially reduced a-l,2-mannosyltransferase activity, as compared to a ciliate that has not been genetically modified, are used to produce the glycoproteins containing predominantly or substantially uniform Man 3 GlcNAc 2 core N- glycans.
  • the reduction or loss of Algl 1 function is due to a genetic modification in an Algl 1 gene locus, including, but not limited to, one or more deletions, mutations or substitutions in a micronuclear Algl 1 gene. In other embodiments, the reduction or loss of Algl 1 function is due to a genetic modification in the Algl 1 gene locus, including, but not limited to, one or more deletions, mutations or substitutions in a macronuclear Algl 1 gene.
  • Genetic modifications suitable for use with the methods described herein include any genetic modifications that inactivate or reduce expression of Algl 1, inactivate or reduce 1 ,2-mannosyltransferase activity of a protein encoded by Algl 1, and/or mislocalize a 1,2-mannosyltransferase protein encoded by Algl 1.
  • one or more inhibitory R As can be used to silence a- 1,2-mannosyltransferase expression or a- 1,2-mannosyltransferase activity by RNA interference (RNAi) alone or in combination with any other methods described herein.
  • RNAi is a process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in cells (Hutvagner and Zamore, 2002, Curr. Opin. Genet. Dev. 12:225-232; Sharp, 2001, Genes Dev. 15:485-490).
  • "Silencing" a target gene means the process whereby a cell containing and/or secreting a certain product of a target gene when not in contact with the agent, will contain and/or secret at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent.
  • Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.
  • RNA agent is an RNA agent, which can down- regulate the expression of a target gene, e.g., a a- 1,2-mannosyltransferase gene.
  • a target gene e.g., a a- 1,2-mannosyltransferase gene.
  • An iRNA agent ⁇ e.g., a double stranded iRNA agent or an small inhibitory RNA agent) may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mR A sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms.
  • RNAi can be used in ciliates (e.g., Tetrahymena spp.) to silence gene expression (Howard-Till and Yao (2006) Mol. Cel. Biol. 26, 8731-8742).
  • expression of a target gene ⁇ e.g., a-l,2-mannosyltransferase gene
  • RNAi technology by targeting nucleotide sequences complementary to any region of the sequence encoding the target protein ⁇ e.g., a-l,2-mannosyltransferase) to form structures that prevent transcription of the gene in target cells.
  • a 400 to 500 base pair length of a target gene can cloned in an inverted orientation around a 90-bp linker containing a 50-bp intron from an unrelated gene.
  • This hairpin construct can then cloned into an rDNA vector under the control of a promoter, for example an inducible MTT promoter.
  • the iRNA agents can be used to silence expression of a- 1,2- mannosyltransferase can comprise about 16 to about 500 nucleotides in each strand, wherein one of the strands contains a region having substantially complementary to, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) complementary ⁇ e.g., having zero or one or more mismatched nucleotides) to a region ⁇ e.g., a transcribed region or a regulatory region) in a- 1 ,2-mannosyltransferase mRNA.
  • iRNA agents can mediate silencing of a gene, e.g., by RNA degradation.
  • RNA is also referred to herein as the RNA to be silenced.
  • a gene is also referred to as a target gene.
  • the RNA to be silenced is aciliate a-l,2-mannosyltransferase gene.
  • the iRNA agent can be a double stranded (ds) iRNA agent.
  • ds iRNA agent is an iRNA agent which includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure.
  • a “strand” herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides).
  • the two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g., by a linker, e.g., a polyethyleneglycol linker, to form but one molecule.
  • At least one strand can include a region which is sufficiently complementary to a target RNA ⁇ e.g., a-l,2-mannosyltransferase mRNA). Such strand is termed the "antisense strand”.
  • a second strand comprised in the dsRNA agent which comprises a region complementary to the antisense strand is termed the "sense strand.”
  • a ds iRNA agent can also be formed from a single RNA molecule which is, at least partly; self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region.
  • the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.
  • the iRNA agents suitable for use with the methods described herein can be delivered to a cell by methods known in the art and as described herein.
  • the iRNA agent can be expressed from an expression vector or the iRNA agent can be delivered directly into a cell by transformation according to any of the methods described herein.
  • the iRNA agents can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from an engineered RNA precursor.
  • the iRNA agents may be designed using methods known in the art (e.g., "The siRNA User Guide,” available at Rockefeller edu/labheads/tuschl/siRNA).
  • the effectiveness of an iRNA agent in silencing expression of a-1,2- mannosyltransferase can be assessed by any of a number of assays following introduction of the into a cell. These assays include, but are not limited to, Western blot analysis using antibodies that recognize the targeted a-l,2-mannosyltransferase gene product following sufficient time for turnover of the endogenous pool after new protein synthesis is repressed, and Northern blot analysis to determine the level of existing target a-l,2-mannosyltransferase mRNA. Negative control iRNAs can be designed to have generally have the same nucleotide composition as the selected iRNA agent, but without significant sequence complementarity to the targeted genome.
  • Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected iRNA agent; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome.
  • negative control iRNAs can be designed by introducing one or more base mismatches into the sequence. Such negative controls are used to, e.g., confirm the specificity of a test iRNA agent.
  • An iRNA agent useful for silencing expression of a- 1,2- mannosyltransferase can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al. (1996), Bioorganic & Medicinal Chemistry 4: 5-23).
  • the genetically modified ciliates can further comprise a transgene for the expression of a recombinant polypeptide of interest (e.g. , a therapeutic polypeptide).
  • Polypeptides encoded by transgenes suitable for expression by the genetically modified ciliates described herein are not limited to any particular protein and can include any polypeptide which it is desirable to produce with predominantly or substantially uniform Man 3 GlcNAc 2 core N-glycans.
  • Any method known in the art can be used to select for the genetically modified ciliates described herein. Suitable methods include, but are not limited to, selection for resistance to an antibiotic. In certain embodiments, phenotypic assortment can also be used to select for one or more genetically modified ciliates suitable for use with the methods described herein (see http://www ciliate.org/genetics.shtml).
  • the genetically modified ciliates having significantly reduced a- 1 ,2- mannosyltransferase activity can also have significantly increased a-l ,2-mannosidase activity, as described above.
  • the genetically-modified ciliates of the invention can be produced by any means of genetic modification known in the art.
  • the ciliates are transformed with an expression vector which expresses a heterologous protein at least transiently, and which may or may not include sequences necessary for autonomous replication.
  • the ciliates are transformed with integration vectors which can cause a gene encoding a heterologous protein to be integrated into either the micronuclear or macronuclear genome. Integration vectors may cause random integration of the desired heterologous sequences, or may promote homologous recombination whereby the
  • heterologous sequences are inserted at a specific desired location in the ciliate genome.
  • the vectors described herein can comprise regulatory elements which are suitable for promoting expression in a ciliate host, or regulatory elements can be supplied by an endogenous gene into which a heterologous coding region integrates. Suitable regulatory regions include, but are not limited to, promoters, enhancers, termination sequences, polyadenylation signals, signal polypeptides and proprotein domains involved in the expression and secretion of proteins. For example, such regulatory elements can provide efficient heterologous expression of heterologous proteins in ciliates under control of promoters and/or terminators which are derived from ciliate genes. Such vectors can comprise naturally occurring promoters and/or terminators from proteins secreted at a high level in ciliates.
  • the promoters and/or terminators can be selected from proteins secreted at a high level independent of the cell-cycle (U.S. Patent Publication No. 2006/0127973; PCT Intl. Pub. WO2003/078566).
  • Inducible promoters from ciliate genes have also been described that allow expression of heterologous genes.
  • heat- inducible promoters of the heat shock protein family of ciliates are also suitable for use with the methods described herein.
  • Suitable heat shock promoters from Tetrahymena spp. are known in the art (see, .e.g., PCT Intl. Pub. WO2007/006812).
  • Such vectors can further comprise a 5' regulatory region from an endogenous ciliate gene containing a promoter region operably joined to the heterologous coding region and/or a 3' regulatory region from the same or a different ciliate gene. Suitable regulatory regions from ciliate genes are well known in the art.
  • Vectors suitable for use with the methods described herein include, but are not limited to: the pXS76 shuttle vector (which can be used for insertion of transgenes downstream of a cadmium-inducible promoter from the MTTl metallothionein gene), rDNA vectors (Tondravi et al. (1986), Proc. Natl. Acad. Sci USA 83:4396; Yu et al. (1989), Proc. Natl. Acad. Sci USA 86: 8487-8491), and high copy number ribosomal DNA vectors (such as pD5H8).
  • the pXS76 shuttle vector which can be used for insertion of transgenes downstream of a cadmium-inducible promoter from the MTTl metallothionein gene
  • rDNA vectors Teondravi et al. (1986), Proc. Natl. Acad. Sci USA 83:4396; Yu et al. (1989), Proc. Natl
  • rDNA-based vectors can be circular vectors containing 5' non- translated sequences comprising two or more ori sequences from a ciliate.
  • One or more nucleic acid fragments containing heterologous coding regions ⁇ e.g., a selectable marker, a- 1 ,2-mannosidase gene, a-l,2-mannosyltransferase knock-out construct, or other transgene) can also be added to the vector using methods known to those skilled in the art.
  • Such vectors can further comprise 5' untranslated regions of a ciliate gene and a 3' untranslated regions of a ciliate gene. These untranslated regions can be inserted upstream and downstream of the selectable marker and/or the transgene.
  • nucleic acid sequences described herein can be cloned using standard cloning procedures in the art, as described by Sambrook et al, eds., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989).
  • a transgene to be expressed in a ciliate such as Tetrahymena
  • additional factors may be considered to enhance protein expression, processing and/or trafficking in a ciliate.
  • Methods for creating mitotically stable ciliate transformants for example, by integration of a heterologous gene by homologous DNA recombination, are known in the art. For example, methods for generating Tetrahymena spp. having targeted gene knockouts by homologous DNA recombination are described in Bruns & Cassidy-Hanley (1999), Methods Cell Biol. 62: 501-512; Hai et al. (1999), Methods Cell Biol. 62:514-531; Gaertig et al. (1999), Nature Biotech. 17:462-465; Cassidy-Hanley et al. (1997), Genetics 146: 135- 147).
  • ciliate cells can be transformed with a homologous recombination vector capable of replacing one or more a- 1,2- mannosyltransferase genes in the micronucleus with a nucleic acid sequence that reduces or abolishes activity or expression of the a-l,2-mannosyltransferase.
  • T. thermophila expresses two major ⁇ -tubulin genes, BTU1 and BTU2, which encode identical ⁇ -tubulin proteins (Gaertig et al. (1993), Cell Mot. Cytoskel. 25:245-253). Either of these two genes (but not both at once) can be disrupted without a detectable change in the cell phenotype.
  • Homologous recombination vectors useful for generating genetically modified ciliates having reduced or abolished a-l,2-mannosyltransferase expression or activity include, but are not limited to knockout cassettes suitable for replacing the micronuclear DNA segment extending from a sequence between the regions flanking all or part of the micronuclear a-l,2-mannosyltransferase locus.
  • Such homologous recombination vectors can further comprise a selection marker such that cells having reduced or abolished a-l,2-mannosyltransferase expression or activity can be selected by growth in a biostatic or biocidal drug.
  • a selection marker such that cells having reduced or abolished a-l,2-mannosyltransferase expression or activity can be selected by growth in a biostatic or biocidal drug.
  • the selection marker enables selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic or biocidal drugs.
  • a heterologous gene that is to be expressed in the cell also may be included within the construct.
  • any of the vectors described herein can be generated using molecular cloning techniques that are well-known in the art. Further, techniques for the procedures of molecular cloning can be found in Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Glover et al.
  • Vectors can be introduced into ciliates using established protocols or any method known to one skilled in the art.
  • transformation of ciliates can be achieved by microinjection (Tondravi and Yao (1986), Proc. Natl. Acad. Sci. USA 83:4369- 4373), electroporation (Gaertig and Gorovsky (1992), Proc. Natl. Acad. Sci. USA 89:9196- 9200), or biolistically (Cassidy-Hanley et al. (1997), Genetics 146: 135-147). Transformation of the either the somatic macronucleus or the generative micronucleus is also possible in ciliates which possess both, including Tetrahymena spp.
  • ciliate cells can be transformed with a chimeric gene by particle bombardment (also known as biolistic transformation) (Cassidy-Hanley et al.
  • Particle bombardment transformation can be achieved by several ways.
  • inert or biologically active particles can be propelled at cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the chimeric gene.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Other variations of particle bombardment now known or hereafter developed, can also be used.
  • Microcarrier bombardment can also be used to transform ciliate cells by means of DNA-loaded gold particles (US 6,087,124; EP 847 444; WO 1998/001572).
  • microcarrier bombardment with DNA-coated gold is used as a means of introducing heterologous genes into ciliates.
  • microcarrier bombardment can be used to transform ciliates and introduce genes into the (germline) micronucleus
  • the vector can further comprise a selectable cassette marker to permit selection for transformed cells (e.g., a neo 2 cassette) (Gaertig et al. (1994), Nucleic Acids Res. 22:5391-5398). Selection of transformants can be achieved by growing the cultured ciliates in a medium which allows only the transformants to survive. Suitable selection agents include antibiotics which will kill most all non-transformants but allow transformants (which also possess an antibiotic resistance gene) to survive. A number of antibiotic-resistance markers are known in the art (Debono and Gordee (1994) Annu. Rev. Microbiol. 48:471-497). Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention.
  • a selectable cassette marker to permit selection for transformed cells.
  • the present invention provides glycoproteins having predominantly Man 3 GlcNAc 2 core N-glycans.
  • a genetically-modified ciliate as described herein, is used to express a heterologous glycoprotein.
  • the glycoproteins are then isolated from the cells or culture medium by methods known in the art.
  • Heterologous proteins produced in ciliates can be targeted to either a constitutive secretory pathway or to a regulated secretory pathway.
  • Ciliates can engage in regulated secretion of proteins stored in cortical secretory organelles (granules) which are discharged in a stimulus-dependent or regulated fashion (17, Miller et al. (1990)).
  • Dense core granules are specialized for stimulus-dependent secretory granules that function in exocytosis in ciliates. In Tetrahymena spp., these dense core granules are termed mucocysts, whereas the dense core granules of Paramecium tetraurelia are termed trichocysts (Hausmann (1978); Rosati and Modeo (2003)).
  • Regulated secretion in ciliates can be triggered by the presence of chemical mediators known as secretagogues.
  • mediators can cause increased levels of intracellular calcium (Ca 2+ ), which, in turn, trigger fusion of cortical granules with the plasma membrane resulting in a release of the granule contents into the surrounding extracellular space.
  • secretagogues useful in the invention include, but are not limited to, dibucaine, alcian blue and Ca 2+ ionophores.
  • mutant strains defective in the release of hydrolytic enzymes have also known in the art (Hunseler et al. (1992), Dev. Genet. 13: 167-173).
  • mutant strains lacking or exhibiting reduced levels of secreted hydrolytic proteases can be used for the production of surface expressed or secreted heterologous polypeptides.
  • a ciliate as described herein can be any free-living ciliate, including but not limited to species in the genera Tetrahymena, Paramecium, Blepharisma, Colpidium, Euplotes, Stylonichia and Oxytricha. In some embodiments, the ciliate is a Tetrahymena spp.
  • the free-living ciliates are a large and diverse phylum (Ciliata) whose members display a structural and functional complexity comparable to that of higher metazoa (Frankel (2000), Meth. Cell Biol. 62: 27-125; Turkewitz et al. (2002), Trends in Genetics, 18, 35-40) and include over 7,000 species with 11 major subdivisions. Tetrahymenids and Paramecium belong to the Oligohymenophoreans.
  • Ciliates suitable for use with the methods described herein include, but are not limited to Tetrahymena thermophila, Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi, Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis, Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes
  • octocarinatus Euplotes vannus, Monoeuplotes crassus, Blepharisma japonicus.
  • the genetically modified ciliates described herein are genetically modified Tetrahymena spp., including T. thermophila.
  • Tetrahymena spp. are amenable to genetic manipulation, can be grown on a large scale and have a doubling time of 1.5-3 hrs. Unlike T. thermophila, which has an optimal growth temperature of 35°C, the optimal growth temperature for T. pyriformis is 34°C. Cells reach high-density in a short time on a variety of inexpensive media and can be expanded for growth in bioreactors up to several thousand liters in size (Hellenbroich et al. (1999); Appl. Microbiol. Biotechnol. 51 :447; de Coninck et al. (2000), J. Industr. Microbiol. Biotechnol. 24:285).
  • Some ciliates exhibit nuclear dimorphism and contain two distinct types of nuclei in each cell.
  • the micronucleus (MIC) is diploid and contains five pairs of transcriptionally inert chromosomes.
  • the macronucleus is polyploid and functions as the transcriptionally active nucleus during vegetative growth.
  • the micronucleus functions as the repository for genetic information for progeny produce by conjugation during sexual reproduction. During conjugation, the micronucleus undergoes meiosis to give rise to two pronuclei that are reciprocally exchanged between sexually mating cells.
  • the micronucleus Upon fusion of the haploid gametes to produce a new zygotic micronucleus, the micronucleus undergoes two post-zygotic divisions resulting in the formation of new micronuclei and macronuclei. Upon formation of new macronuclei, the old macronuclei are resorbed and not transmitted to the sexual progeny. The cells then reproduce by asexual until the next round of conjugation.
  • MAC macronucleus
  • IESs internal eliminated sequences
  • CBS chromosome breakage sequence
  • the present invention provides compositions comprising a population of glycoproteins having predominantly or substantially uniform Man 3 GlcNAc 2 core N-glycans.
  • the advantages of producing glycoprotein compositions having a predominantly Man 3 GlcNAc 2 core glycan include reduced presence of undesired glycoforms, less heterogeneous mixtures of glycoproteins, and/or higher concentrations of the more effective glycoform(s). Therefore, a pharmaceutical composition comprising glycoproteins having predominantly Man 3 GlcNAc 2 core N-glycan can have beneficial features, including but not limited to, reduced immunogenicity, greater manufacturing consistency, and higher efficacy/potency.
  • a glycoprotein of the present invention comprises at least one Man 3 GlcNAc 2 core N-glycan structure at one or more Asparagine residues within the polypeptide.
  • the glycoproteins are produced in a host cell that has been genetically engineered to produce a population of glycoproteins having Man 3 GlcNAc 2 as the
  • the genetically modified ciliates are transformed with one or more nucleic acids encoding the desired recombinant glycoprotein.
  • the recombinant glycoprotein encoded by the nucleic acid is a therapeutic polypeptide (e.g., a cytokine, peptide hormone or antibody).
  • the genetically modified ciliates described herein are incubated under conditions suitable for producing the polypeptide.
  • the ciliates can secrete the recombinant polypeptide into the culture medium either through a constitutive secretory pathway or a regulated secretory pathway.
  • the recombinant glycoprotein can then be separated from other components of the culture medium and be resuspended in a suitable vehicle to make the compositions.
  • glycoproteins are produced in a host cell that has been genetically engineered to produce glycoproteins having predominantly or substantially uniform
  • the immunoglobulin molecule can comprise at least one
  • the Man 3 GlcNAc 2 glycan structure is on each Asn-297 of each C R 2 region in a dimerized immunoglobulin.
  • the present invention provides compositions comprising immunoglobulins which are predominantly glycosylated with an N-glycan consisting essentially of Man3GlcNAc 2 glycan structure at Asn-297.
  • one or more carbohydrate moieties found on an immunoglobulin molecule may be deleted and/or added to the molecule, thus adding or deleting the number of glycosylation sites on an immunoglobulin.
  • the position of the N-linked glycosylation site within the C H 2 region of an immunoglobulin molecule can be varied by introducing asparagines (Asn) or N-glycosylation sites at varying locations within the molecule.
  • Asn-297 is the N-glycosylation site typically found in murine and human IgG molecules (Kabat et al., Sequences of Proteins of Immunological Interest, 1991), the methods describe herein are not limited to Man3GlcNAc 2 glycan structure at this site, nor does this site necessarily have to be maintained for function.
  • Glycosylation of the Fab region of an immunoglobulin can also occur in serum antibodies, for example at Asn-75 (Rademacher et al. (1986), Biochem. Soc. Symp. 51 : 131-148). Glycosylation in the Fab region of an immunoglobulin molecule is an additional site that can be combined in conjunction with N-glycosylation in the Fc region, or alone.
  • a nucleic acid encoding the heavy and light chains of the immunoglobulin can be synthesized using overlapping oligonucleotides and each separately cloned into an expression vector for expression in a host cell.
  • the recombinant immunoglobulin encoded by the nucleic acid can be a humanized immunoglobulin.
  • the site for the N-glycan linkage can be at an asparagine at a different site within the immunoglobulin molecule (other than Asn-297), or in combination with the N-glycosylation site in the Fab region.
  • antibody is used in the broadest sense and includes single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions which will bind to multiple epitopes or antigens.
  • the terms specifically cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they contain or are modified to contain at least the portion of the C H 2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the C R 2 domain, or a variant thereof. Included within the term are molecules comprising the Fc region, Fc fusions and antibody- like molecules.
  • these terms can refer to an antibody fragment of at least the Fab region that at least contains an N-linked glycosylation site.
  • antibody classes of Igs, namely, IgG, IgA, IgE, IgM, and IgD.
  • subtypes of IgGs namely, IgGl, IgG2, IgG3 and IgG4.
  • Fc refers to the ' fragment crystallized ' C-terminal region of the antibody containing the C R 2 and C R 3 domains.
  • Fab refers to the 'fragment antigen binding' region of the antibody containing the VH, CHI, VL and CL domains.
  • mAb monoclonal antibody
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a first species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they contain or are modified to contain at least one C R 2.
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a first species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they contain or are modified
  • “Humanized” forms of non-human ⁇ e.g., murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 , or other antigen-binding subsequences of antibodies) which contain sequences derived from human immunoglobulins.
  • An Fv fragment of an antibody is the smallest unit of the antibody that retains the binding characteristics and specificity of the whole molecule.
  • the Fv fragment is a noncovalently associated heterodimer of the variable domains of the antibody heavy chain and light chain.
  • the F(ab)'2 fragment is a fragment containing both arms of Fab fragments linked by the disulfide bridges.
  • Polypeptides having a Man 3 GlcNAc 2 N-glycan structure as described herein can also comprise fusion polypeptides.
  • Polypeptides produced by the genetically modified ciliates described herein can further include two or more polypeptides attached to one another by recombinant means.
  • the polypeptide can be a fusion between a cytokine and an antibody.
  • a cytokine e.g., an antibody
  • fusion protein can further comprise a flexible linker sequence (for example a 5 -10 amino acid sequence composed primarily of alanine and glycine residues) such that the biological activity and folding of the fused polypeptides is maintained.
  • fusion of a polypeptide to the C-terminus of the full length antibody can be useful.
  • N and C terminal fusions are also possible in certain circumstances, for example where the fusion comprises a single chain antibody fragment (scFv).
  • Such fusion polypeptides can present combinations of biological activity and function that are not found in nature to create novel and useful molecules of therapeutic and industrial applications.
  • Biological activities of interest include, but are not limited to, enzymatic activity, receptor and/or ligand activity, immunogenic motifs, and structural domains.
  • Such fusion polypeptides are well known in the art, and the methods of creation will be well-known to those in the art.
  • Polypeptides having a Man 3 GlcNAc 2 N-glycan structure as described herein can be variants of native polypeptides, wherein a fragment of the native polypeptide is used in place of the full length native polypeptide.
  • pre-pro-, and pre-peptides can also be used in conjugation with the methods described herein.
  • Variant polypeptides may be smaller in size that the native polypeptide, and may comprise one or more domains of a larger polypeptide. Also included are truncations of the polypeptide and internal deletions which may enhance the desired therapeutic effect of the polypeptide. Any such forms of a polypeptide is contemplated to be useful in the present invention provided that the desired biological activity of the polypeptide is preserved.
  • compositions having Man 3 GlcNAc 2 as the predominant N-glycan can be incorporated into pharmaceutical compositions wherein the polypeptide is an active therapeutic agent (See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980).
  • the composition can depend on the intended mode of administration and therapeutic application.
  • the composition can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination.
  • compositions or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions for parenteral administration can be sterile, substantially isotonic, pyrogen-free, sterile, and prepared in accordance with GMP of the U.S. Food and Drug Administration or similar body.
  • the compositions can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • the compositions can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (See Langer (1990), Science 249: 1527 and Hanes (1997), Advanced Drug Delivery Reviews 28: 97-119.
  • Polypeptides or antibodies having a Man 3 GlcNAc 2 N-glycan structure produced according to the methods described herein can be incorporated into pharmaceutical compositions comprising the polypeptide as an active therapeutic agent and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The form can depend on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent can be selected so as not to affect the biological activity of the combination.
  • compositions or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • polypeptides having a Man 3 GlcNAc 2 N-glycan structure as the predominant N-glycan can also be incorporated into a variety of diagnostic kits and other diagnostic products such as an array.
  • the polypeptides having a Man 3 GlcNAc 2 N-glycan structure as the predominant N-glycan can have many therapeutic applications for indications including, but not limited to, cancers, inflammatory diseases, infections, immune diseases, and autoimmune diseases.
  • Example 1 Expressing a-l,2-mannosidase in a Ciliate
  • Man 3 GlcNAc 2 glycan is native to T. thermophila and other ciliates and therefore is not deleterious to engineered cells.
  • Man 3 GlcNAc 2 provides a native glycan platform for complex glycan synthesis in ciliates that is quite distinct from the non-native MansGlcNAc 2 glycan generated in previous efforts in yeast.
  • yeast which, contain a cell wall composed of a mixture of carbohydrates and proteoglycans including ⁇ 1 ,3 and ⁇ 1 ,4 glucans, mannoproteins and chitin, and which is essential to the structural integrity of the cell
  • expression of heterologous a-l,2-mannosidase does not adversely affect the health of the cell.
  • the removal of Ochl activity leads to a substantial loss of cell wall mannose content in the form of mannoproteins that results in a structurally compromised cell. This is evidenced by characteristic cell- wall deficiency phenotypes in Ochl mutants of a number of different yeast species such as cell-aggregation, bloating and slow growth.
  • protein expression can be severely compromised in a fungal organism with this genetic background.
  • the methods described herein relate to ciliate cell lines that produce uniform Man 3 GlcNAc 2 glycans that are wild-type except for expression of a heterologous a-l,2-mannosidase.
  • An expression cassette was designed to express an a- 1,2- mannosidase (GenBank Gene ID, 3408991) from the ciliate Entamoeba histolytica, in the ciliate Tetrahymena thermophila ( Figure 6).
  • the expression cassette contains a
  • cycloheximide resistance cassette that incorporates the rpl29 gene and a promoter derived from the Tetrahymena thermophila catalase gene (GenBank Gene ID, 7837249) upstream of the E. histolytica a-l,2-mannosidase.
  • the expression cassette is flanked by approximately 1Kb of DNA homologous to the Tetrahymena BTU1 gene locus to direct homologous recombination at that locus.
  • the 3' BTU targeting DNA also acts as a terminator for expression of the a-l,2-mannosidase gene.
  • Example 2 Alg 11 Deficient Ciliates
  • Uniform Man 3 GlcNAc 2 glycans can also be produced from ciliates by generating a strain that is deficient in the Algl 1 gene product.
  • Algl 1 is an a- 1,2- mannosyltransferase that sequentially adds a 1 ,2 linked mannose residues to the lipid-linked glycan precursor on the cytoplasmic face of the ER.
  • the lipid-linked MansGlcNAc 2 glycan precursor is then translocated to the luminal face of the ER where three glucose residues are added sequentially to the terminal a 1 ,2 linked mannose residue prior to transfer to protein.
  • Algl 1 is an essential gene in a majority of eukaryotic cells. Since ciliates, such as Tetrahymena have a ploidy of 45 gene copies in their macronucleus, generating a strain that is deficient in the Algl 1 gene product can be used as a strategy to produce uniform Man 3 GlcNAc 2 glycans. In ciliates, phenotypic assortment following introduction of a disruption cassette can result in a gene dosage effect where a majority of glycans are produced as Man 3 GlcNAc2 structures.
  • the Tetrahymena macronucleus has a ploidy of 45 therefore in the context of a knockout, to ensure that the native locus has been completely replaced by the knockout cassette, cells can be "pushed" in increasing levels of drug so that the higher the tolerence to drug the more copies of the knockout cassette containing the drug resistance marker have replaced the native allele.
  • cells can be pushed by increasing levels of selective pressure ⁇ e.g., the concentration of a selective drug).
  • Cells phenotypically assorted in increasing concentrations of selection drug exhibited a slow growth rate phenotype, indicating of the importance of this gene to cell health.
  • Any approach known in the art that is suitable for reducing, or eliminating Algl 1 function in a ciliate can be used in conjunction with the methods described herein.
  • An exemplary approach includes, but is not limited to, introducing random mutations to cells containing an Algl 1 knockout allele(s). Strains of such cells can then be screened to identify strains that have increased tolerance for the knockout, are healthier or produce a larger relative pool of Man 3 GlcNAc 2 glycans.
  • Man 3 GlcNAc 2 glycans in genetically modified fungal microorganisms that normally produce Glc 3 Man9GlcNAc 2 N- glycan precursors.
  • Late stages of N-glycan precursor synthesis can be interrupted through deletion of the ALG3 gene to keep N-glycan precursor intermediates from becoming larger than Man 5 GlcNAc 2 .
  • this approach can be performed in a strain background that additionally does not produce N-glycan outer chain mannan in the Golgi.
  • Such strains that do not produce N-glycan outer chain mannan in the Golgi can be generated by deletion of ochl.
  • the Man 5 GlcNAc 2 glycan can become transferred to proteins and converted to a Man 3 GlcNAc 2 glycan through the action of an in vivo expressed a 1,2- mannosidase.
  • This methodology can be applied to generating Man 3 GlcNAc 2 in fungi, however results can vary from organism to organism. In contrast to fungal glycoengineering, ciliate cells naturally lack an ALG3 gene and produce and process Man 5 GlcNAc 2 to
  • the Algl 1 gene in Tetrahymena encodes a a 1,2- mannosyltransferase.
  • Algl 1 deficient strains will be generated to eliminate a- 1,2- mannosyltransferase thereby generating a genetically modified ciliate predominantly expressing polypeptides or antibodies having a Man 3 GlcNAc 2 N-glycan structure.
  • a a 1 ,2-mannosidase can further be expressed on the ER of such proteins to enhance the ability of a ciliate to form Man 3 GlcNAc 2 precursors.
  • thermophila strains were cultured in 100 ml Neff (5% glucose, 2.5 g/L Bactopeptone, 2.5 g/L yeast extract). Spent culture medium (SCM) was collected by centrifugation at 3500 x g for 10 minutes and then concentrated 100 fold in Vivaspin 20 columns (Sartorius) containing a 30 kDa molecular weight cut-off (MWCO) membrane. Concentrated SCM was washed with 10 mM Tris-Cl pH 8.0 in the same columns and sample proteins were purified by anion-exchange chromatography.
  • Neff 5% glucose, 2.5 g/L Bactopeptone, 2.5 g/L yeast extract.
  • SCM Spent culture medium
  • Vivaspin 20 columns Sartorius
  • MWCO molecular weight cut-off
  • N-Glycosidase F N-Glycosidase F
  • SDS SDS-Sepse F
  • HPAEC high-pressure anion exchange chromatography
  • the a-l,2-mannosidase expression cassette copy number can be increased.
  • a more efficient native promoter or a more efficient heterologous promoters can be used in conjunction with the methods described herein.
  • the methods described herein can be performed using a more active a-l,2-mannosidase gene.

Abstract

The invention is directed to methods for recombinant glycoprotein production and, in particular, genetically modified ciliates expressing glycoproteins having predominantly Man3GlcNAc2 core N-glycan structures and methods and products related to the production of recombinant glycoproteins with predominantly Man3GlcNAc2 core N-glycan structures.

Description

PRODUCTION OF GLYCOPROTEINS IN
GENETICALLY MODIFIED CILIATES
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/316,168, filed on March 22, 2010, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to recombinant protein production and, in particular, methods and compositions for the production of recombinant glycoproteins in ciliates.
BACKGROUND OF THE INVENTION
[0003] Recombinant proteins are important in a wide range of applications that extend from alternative fuel production to the treatment of human and animal diseases. The manufacture of genetically engineered enzymes, therapeutic proteins, vaccines and biopolymers constitutes a multibillion dollar-per-year industry, independent of the large market for recombinant proteins produced in basic research (Pavlou and Reichert (2004), Nat. Biotechnol. 22: 1513-1519; Langer, 3rd Annual Report and Survey of Biopharmaceutical Manufacturing, Capacity and Production, BioPlan Associates, Inc. 2005).
[0004] Secreted glycoproteins, in particular, represent the fastest growing set of therapeutically relevant recombinant proteins (Sethuraman and Stadheim (2006), Curr. Opin. Biotechnol. 17:341-346; Jefferis (2005), Biotechnol. Prog. 21 : 11-16; Logtenberg (2007), Trends Biotechnol. 25:390-394). The production of biologically functional secreted glycoproteins is dependent on the coordination of correct protein folding and the addition of appropriate carbohydrate modifications. One important class of protein modification is asparagine (N)-linked glycosylation, which involves the addition of a glycan moiety to an asparagine residue that resides in the following consensus sequence: Asn-X-Ser/Thr, where X can be any amino acid except proline (Kornfeld and Kornfeld (1985), Rev. Biochem. 54:631- 664). N-linked glycosylation can play an essential role in many different facets of glycoprotein production and function, including correct protein folding, efficient trafficking through the secretory pathway, protein stability, and biological or therapeutic activity (Fan et al. (1997), Eur. J. Biochem. 246:243-251; Sitia and Braakman (2003), Nature 426:891-894; Skropeta (2009), Bioorg. Med. Chem. 17:2645-2653; Rudd et al. (2001), Science 291 :2370- 2376; Helenius and Aebi (2001), Science 291 :2364-2369). In the case of protein therapeutics, glycosylation can significantly affect the half-life of the drug in the bloodstream and, thus, therapeutic efficacy (Sinclair and Elliot (2005), J. Pharm. Sci. 94: 1626-1635;
Hoffmeister et al. (2003), Science 301 : 1531-1534). Additionally, the composition of the N- glycans of monoclonal antibodies, the largest group of protein therapeutics, can play a critical role in the antibody's ability to recruit host immune system effector cells through the processes of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Shantha Raju (2008), Curr. Opin. Immun. 20:471-478; Nimmerjahn and Ravetch (2008), Nat. Rev. Immunol. 8:34-47; Jeffries and Lund (2002), Immunol. Lett. 82:57- 65; Mimura et al. (2001), J. Biol. Chem. 276:45539-45547).
[0005] There is a need in the art, therefore, for methods for controlling the composition and uniformity of N-linked glycosylation of proteins produced by recombinant cell lines. In particular, there is a need for methods of engineering cell lines which can produce large amounts of glycoproteins with substantially uniform glycosylation which is identical or sufficiently similar to human or mammalian glycosylation to be useful in therapeutic applications. This invention addresses these needs.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans.
[0007] In another aspect, the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans, wherein the ciliates have significantly increased a-l,2-mannosidase activity.
[0008] In still a further aspect, the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans, wherein the ciliates express a heterologous sequence encoding a protein having a- 1 ,2-mannosidase activity.
[0009] In some embodiments, the protein having a-l,2-mannosidase activity comprises an endoplasmic reticulum targeting sequence.
[0010] In some embodiments, the protein having a-l,2-mannosidase activity comprises a signal sequence. [0011] In some particular embodiments, the protein having a-l,2-mannosidase activity comprises a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 17.
[0012] In some embodiments, the protein having a-l,2-mannosidase activity is selected from the group consisting of Aspergillus saitoi a-l,2-mannosidase, Trichoderma reesei a-l,2-mannosidase, Penicillium citrinum a-l,2-mannosidase, Aspergillus nidulans a- 1 ,2-mannosidase, Homo sapiens a-l,2-mannosidase I A, Homo sapiens IB a- 1,2- mannosidase, Lepidopteran insect Type I a-l,2-mannosidase, Homo sapiens a D
mannosidase, Xanthomonas a-l,2-mannosidase, mouse IB a-l,2-mannosidase, and Bacillus sp. a-l,2-mannosidase.
[0013] In yet another aspect, the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans, wherein the ciliates have significantly decreased a-l,2-mannosyltransferase activity.
[0014] In still a further aspect, the invention described herein relates to genetically modified ciliates that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans, wherein the ciliates have a micronuclear genotype comprising one or more nonfunctional Algl 1 alleles.
[0015] In some embodiments, the genetic modification is selected from the group consisting of a deletion, an insertion, a substitution or an inversion in an Algl 1 allele.
[0016] In certain embodiments, the genetically modified ciliates are selected from the group consisting of Tetrahymena thermophila, Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi, Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis, Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes octocarinatus, Euplotes vannus, Monoeuplotes crassus, and Blepharisma japonicus.
[0017] In yet another aspect, the invention described herein relates to a method for producing a recombinant glycoprotein having predominantly Man3GlcNAc2 core N- glycans, the method comprising: (a) transforming ciliates with an expression construct encoding the recombinant glycoprotein, (b) culturing the ciliates under conditions which promote expression of the recombinant glycoprotein, and (c) isolating the recombinant glycoprotein. [0018] In some embodiments, the recombinant glycoprotein is a human protein. In some embodiments, the recombinant glycoprotein is a therapeutic protein. In some embodiments, the recombinant glycoprotein is a cytokine. In other embodiments, the recombinant glycoprotein is a fusion protein. In still other embodiments, the recombinant glycoprotein is an antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0019] Figure 1 is a schematic representation of the N-glycan precursor biosynthetic pathways common to many eukaryotes, including mammals and yeast, leading to the assembly of the complete glycan precursor on the dolichol lipid carrier that is transferred to a protein. Genes involved in N-glycan processing are shown by each arrow. Following transfer to the protein, the Glc3Man9GlcNAc2 can be trimmed to remove the three glucose residues in the glycoprotin quality control pathway. A mannose can also be removed by an a-l,2-mannosidase to yield MangGlcNAc2 prior to exit from the ER and further trafficking to the Golgi.
[0020] Figure 2 is a schematic representation of the maturation of yeast
Man8GlcNAc2 N-glycans in the Golgi, which produces high-mannose glycans. Genes involved in N-glycan processing are shown above each arrow.
[0021] Figure 3 is a schematic representation of the maturation of mammalian Man8GlcNAc2 N-glycans in the Golgi, which produces complex glycans. Genes involved in N-glycan processing are shown above each arrow.
[0022] Figure 4 is a schematic representation of predicted ciliate N-glycan precursor biosynthetic pathway. Genes involved in N-glycan processing are shown above each arrow.
[0023] Figure 5 is a schematic representation of the precursor glycans produced in all fungi, plants and animals (Glc3Man9GlcNAc2) and those produced in ciliates
(Glc3Man5GlcNAc2).
[0024] Figure 6 is a schematic representation of the design of an expression cassette used for the expression of a-l,2-mannosidases in a ciliate, Tetrahymena. DNA targeting sequences (5' BTUl and 3' BTUl) were used to direct integration of the cassette at the Tetrahymena BTUl locus. Also shown is a cycloheximide resistance cassette (Cycr) in relation to the Catalase promoter (pr) and the a-l,2-mannosidase gene. In this example, the 3' BTU1 targeting DNA provides a terminator for the expression of the a-l,2-mannosidase gene.
[0025] Figure 7 shows results of experiments in which the a-l,2-mannosidase from Entamoeba histolytica was expressed in a ciliate. A. E. histolytica a-l,2-mannosidase was expressed in Tetrahymena. Tetrahymena cell lysates were prepared from a strain containing an integrated a-l,2-mannosidase expression cassette. Anti-HA western blot analysis indicates that transgenic strains express the recombinant protein. No signal is detected in wild-type cells (WT). B. Recombinant E. histolytica a-l,2-mannosidase is localized in the Tetrahymena endoplasmic reticulum (ER). Immunofluorescence was performed on Tetrahymena transgenic strains using anti-HA antibody directed to an engineered epitope on the recombinant mannosidase. Top left panel shows Tetrahymena nuclei only (DAPI staining). Top right panel shows anti-HA detection of mannosidase.
Bottom left panel shows a merged image of the top two panels. Staining patterns are indicative of an ER localized protein.
[0026] Figure 8 shows results of experiments in which a-l,2-mannosidase significantly increases the relative amount of Man3GlcNAc2 glycans attached to
glycoproteins when expressed in Tetrahymena. Mature glycoproteins from Tetrahymena wild-type strains and transgenic strains expressing a-l,2-mannosidase were purified from spent culture medium. N-linked glycans were released from purified proteins by treatment with PNGaseF and analyzed by high-pressure anion exchange chromatography. Peaks in chromatograms are labeled in red and corresponding structures are shown in figure inserts. The relative amounts of each glycan are shown circled in red. In wild-type cells the total amount of Man3GlcNAc2 glycan isolated represents approximately 44% total glycan. In transgenic strains Man3GlcNAc2 glycan represents approximately 82% total glycan.
[0027] Figure 9 shows results of experiments in which attenuation of Algl 1 function in Tetrahymena results in an increase in relative Man3GlcNAc2 glycan associated with glycoprotein. Mature glycoproteins from a Tetrahymena strain that contains disrupted alleles of the Algl 1 gene were purified from spent culture medium. N-linked glycans were released from purified proteins by treatment with PNGaseF and analyzed by high-pressure anion exchange chromatography. Peaks are labeled in red and corresponding structures are shown in the figure insert. The relative amounts of each glycan are shown circled in red. The results indicate that down-regulation of Algl 1 function results in a relative increase in Man3GlcNAc2 glycan (approximately 71%) attached to protein compared to wild-type cells (approximately 44%; see Figure 8).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The glycosylation of a recombinant protein can vary depending upon the expression system used, and the particular conditions used to produce the given protein. Controlling the type and extent of protein glycosylation can be important for the expression of functional recombinant proteins. Production of polypeptides in non-human sources can result in non-human glycosylation of the protein and, thus, immunogenic responses if the protein is administered to a human subject. For example, hyper-mannosylation in yeast, a- 1,3-fucose and PP-l,2-xylose in plants, N-glycolylneuraminic acid in Chinese hamster ovary cells, and Gal a- 1,3 Gal glycosylation in mice, all cause problems of immunogenicity in humans (Ballou (1990), Methods Enzymol. 185:440-470; Cabanes-Macheteau et al. (1999), Glycobiology 9:365-372; Noguchi et al. (1995), J. Biochem. 117:59-62; Borrebaeck (1993), Immun. Today 14:477-479). Cell culture conditions can also render polypeptide
compositions immunogenic (Patel et al. (1992), Biochem. J. 285:839-845).
[0029] In the case of antibody production, differential glycoforms can also result in pharmacokinetic and pharmacodynamic problems including, but no limited to, issues relating to receptor-interaction, tissue-specific targeting, protease resistance, serum half life, and complement binding (Graddis et al. (2002), Curr. Pharm. Biotechnol. 3:285-297; Jefferis and Lund (1997), Antibody Eng. Chem. Immunol. 65: 111-128; Wright and Morrison (1997), Trends Biotechnol. 15:26-32).
[0030] In general, the methods and compositions described herein relate to methods for generating genetically modified ciliates {e.g., Tetrahymena thermophila) that are capable of substantially uniform glycosylation with glycans comprising distinct carbohydrate structures. In some embodiments, the ciliates have a genetically modified glycosylation pathway such that recombinant glycoproteins expressed in these cells display predominantly or substantially uniformly a Man3GlcNAc2 core N-glycan structure.
[0031] In certain aspects, these genetically modified ciliates can be obtained using a combination of genetic engineering and/or selection of strains which have reduced a- 1,2- mannosyltransferase and/or increased a-l ,2-mannosidase activity than a ciliate that has not been genetically modified.
[0032] In another aspect, the methods described herein provide a method for producing one or more glycoproteins containing predominantly Man3GlcNAc2 core N- glycans. In another aspect, the methods described herein provide a method for producing one or more glycoproteins containing substantially uniform Man3GlcNAc2 core N-glycans. In general, the methods described herein relate to the use of genetically modified ciliates as an expression system for producing glycoproteins, wherein the genetically modified ciliates comprise one or more modifications of the glycosylation pathway such that all, or a predominant molar ratio, of the glycoproteins produced by the ciliate will have
Man3GlcNAc2 core N-glycans.
[0033] The methods and compositions described herein are based in part on the finding that increased a-l ,2-mannosidase activity and/or reduced a-l ,2-mannosyltransferase activity in a ciliate will result in all, or a predominant molar ratio, of the glycoproteins produced by the ciliate having Man3GlcNAc2 core N-glycans.
[0034] The genetically modified ciliates described herein can comprise one or more deletions or disruptions of one or more copies of an Algl 1 gene locus. In one embodiment, deletion or disruption of the Algl 1 gene locus results in the generation of a genetically modified ciliate capable of producing a recombinant polypeptide (e.g. , a therapeutic polypeptide or an antibody) in which Man3GlcNAc2 is the predominant core N- glycan. In other embodiments, a ciliate comprising one or more deletions or disruptions of an Algl 1 gene can be further genetically modified so as to have greater a-l ,2-mannosidase activity than a ciliate that has not been genetically modified.
[0035] In one embodiment, the genetically modified ciliates described herein express a transgene encoding a-l ,2-mannosidase, wherein expression of the a- 1 ,2- mannosidase causes the ciliate to produce glycoproteins having substantially uniform
Man3GlcNAc2 core N-glycans. In another embodiment, the genetically modified ciliates described herein express a transgene encoding a-l ,2-mannosidase and wherein expression of the a-l ,2-mannosidase causes the ciliate to produce glycoproteins having predominantly Man3GlcNAc2 core N-glycans. [0036] In a further embodiment, the genetically modified ciliates described herein comprise at least one modified Algl 1 gene locus which causes reduced a- 1,2- mannosyltransferase activity in the cell, thereby causing the ciliate to produce glycoproteins having predominantly Man3GlcNAc2 core N-glycans. In still further embodiments, the genetically modified ciliates described herein have at least one modified Algl 1 gene locus causing reduced a-l,2-mannosyltransferase activity, or complete loss of a- 1,2- mannosyltransferase activity in the cell, thereby causing the ciliates to produce glycoproteins having substantially uniform Man3GlcNAc2 core N-glycans.
[0037] In still a further aspect, the invention provides for glycoproteins produced by the genetically modified ciliates described herein. In one embodiment, the glycoprotein is a therapeutic polypeptide. In another embodiment, the glycoprotein is an antibody.
[0038] Definitions
[0039] All scientific and technical terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In addition, in order to more clearly and concisely describe the subject matter which is the invention, the following abbreviations and definitions are provided for certain terms which are used in the specification and appended claims.
[0040] As used herein, the following abbreviations have the following meanings:
Glc = glucose,
GlcN =- glucosamine,
GlcNAc = N-acetylglucosaminyl,
Gal = galactose,
GalN = galactosamine,
GalNAc = N-acetylgalactosamine,
Man = mannose,
ManN = mannosamine,
ManNAc = N-acetylmannosamine,
Sia = sialic acid, Neu5AC = N-acetylneuraminic acid (NANA),
Neu5Gc = N-glycolylneuraminic acid,
Fuc = fucose,
GlcA = glucuronic acid, and
IdoA - iduronic acid.
[0041] As used herein, the term "ciliate" means a eukaryote belonging to the kingdom Chromalveolata, the superphylum Alveolata, and the phylum Ciliophora. Ciliates are complex protozoa characterized by the presence of cilia on their cell surfaces and dimorphic nuclei consisting of a macronucleus and one or more micronuclei.
[0042] As used herein, Tetrahymena spp." refers to ciliate protozoa in the family of Tetrahymenidae. Exemplary Tetrahymena spp. include but are not limited to,
T. thermophila and T. pyriformis.
[0043] As used herein, the term "N-glycan" refers to an N-linked oligosaccharide, including, for example, one that is or was attached by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a polypeptide.
[0044] N-glycans can differ with respect to the number of branches (antennae) comprising peripheral sugars {e.g., GlcNAc, Gal, Fuc, Sia) that are added to the
Man3GlcNAc2 structure. N-glycans can be classified according to their branched constituents {e.g., high mannose, complex or hybrid). For example, a "high mannose" type N-glycan can have five or more mannose residues. A "complex" type N-glycan can have at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a Man3GlcNAc2 core. Complex N-glycans can also have Gal or GalNAc residues that can be further modified with Sia or derivatives {e.g., NeuAc). The various N- glycans are also referred to as "glycoforms."
[0045] As used herein, the terms "core N-glycan" means (1) any glycan structure, comprising a multiplicity of saccharide residues with stereospecific linkages, that is transferred from a dolichol pyrophosphate (Dol-PP) glycan precursor to a nascent
glycoprotein (an "initial core N-glycan"), or (2) any glycan structure or substructure in a mature glycoprotein, comprising a multiplicity of saccharide residues with stereospecific linkages, that is present in the initial core glycan (a "processed core N-glycan"). Thus, the structure of a processed core N-glycan includes any oligosaccharide residues (with the same stereospecific linkages) which are found in the initial core N-glycan, but excludes any oligosaccharide residues which were added or removed during glycosyl processing subsequent to transfer of the initial core N-glycan from the Dol-PP glycan precursor.
Referring to Figure 1 , the Glc3Man9GlcNAc2 glycan is the initial core N-glycan for yeast and mammals. Referring to Figure 2 and 3, the processed core N-glycans of yeast and mammals include the Man8GlcNAc2 and Man3GlcNAc2 glycan substructures shown after steps Y4 and M10, respectively. Referring to Figure 4, the Glc3Man5GlcNAc2 glycan is the initial core M- glycan in ciliates. Referring to Figure 8, the processed core N-glycans of ciliates include the Man5GlcNAc2, Man4GlcNAc2 and Man3GlcNAc2 glycan substructures identified
experimentally.
[0046] As used herein, the term "operably joined" refers to a covalent and functional linkage of one or more genetic regulatory elements and one or more genetic coding regions which can cause the coding region(s) to be transcribed into mRNA by an RNA polymerase which can bind to one or more of the regulatory elements. Thus, a regulatory region, including regulatory elements such as a promoter and/or enhancer, is operably joined to a coding region when an RNA polymerase is capable of binding under permissive conditions to a promoter within the regulatory region and causing transcription of the coding region(s) into mRNA. In this context, permissive conditions would include standard intracellular conditions for constitutive promoters, standard conditions and the absence of a repressor or the presence of an inducer for repressible/inducible promoters, and appropriate in vitro conditions, as known in the art, for in vitro transcription systems.
[0047] As used herein, the term "heterologous" means, with respect to two or more genetic or protein sequences, that the sequences do not occur in the same physical relation to each other in nature and/or do not naturally occur within the same genome or protein. For example, a genetic construct may include a coding sequence which is operably joined to one or more regulatory sequences, or to one or more other coding sequences, and these sequences are considered heterologous to each other if they are not operably joined in nature and/or they are not found in the same relation in a genome in nature. Similarly, a protein may include a first polypeptide sequence which is joined by a standard polypeptide bond to a second polypeptide sequence, and these sequences are considered heterologous to each other if they are not found in the same relation in any protein or proteome in nature. [0048] As used herein, the term "recombinant" means, with respect to a protein, that the protein comprises two or more heterologous amino acid sequences, which are encoded by a DNA molecule (e.g. , a plasmid or a chromosome) comprising two or more heterologous nucleic acid sequences, or that the protein is heterologous to the cell in which it is produced. As used herein with respect to a cell, the term "recombinant" means that the cell includes a DNA molecule (e.g., a plasmid or a chromosome) comprising at least one heterologous nucleic acid sequence.
[0049] As used herein, the term "promoter" means a nucleotide sequence which is capable of binding an R A polymerase and initiating transcription of a downstream or 3' coding sequence.
[0050] As used herein, the term "selectable marker" means any genetic sequence which, when expressed, has a biochemical or phenotypic effect which is dominant and selectable by the presence or absence of a selection agent or by physial means (e.g., phenotypic sorting). Selectable marker genes that confer resistance or tolerance to a normally toxic selection agent cause only successfully transfected cells to survive in the presence of the selection agent and are referred to as positive selection markers. Selectable marker genes that confer sensitivity or susceptibility to a normally non-toxic selection agent cause only successfully transfected cells to die in the presence of the selection agent are referred to as negative selection markers. Phenotypic selectable marker genes permit selection based upon morphological or biochemical traits rather than cell death or survival. In some cases, the phenotypic marker is detectable only in the presence of an additional selection agent (e.g., a chromogenic substrate for an enzyme).
[0051] As used herein, the term "vector" means any genetic construct, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of transferring nucleic acids into or between cells. Vectors may be capable of one or more of replication, expression, and insertion or integration, but need not possess each of these capabilities. Thus, the term includes cloning, expression, homologous recombination, and knock-out vectors.
[0052] As used herein, the term "expression vector" means a vector comprising regulatory elements operably joined to one or more coding regions to be expressed in a host cell transformed with the expression vector. [0053] As used herein, the term "transform" means to introduce into a cell an exogenous nucleic acid or nucleic acid analog that encodes a polypeptide sequence which is expressed in that cell (with or without integration into the genome of the cell), and/or that is integrated into the genome of that cell so as to affect the expression of a genetic locus already present within the genome. The term "transform" is used to embrace all of the various methods of introducing such nucleic acids or nucleic acid analogs, including, but not limited to the methods referred to in the art as transformation, transfection, transduction, or gene transfer, and including techniques such as microinjection, DEAE-dextran-mediated endocytosis, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, ballistic injection, viral-mediated transfection, particle bombardment and the like.
[0054] As used herein, the term "polypeptide" means an oligopeptide, polypeptide, or protein. The term "polypeptide" herein can refer to a polypeptide either with and without glycosylation.
[0055] As used herein, the term "glycopolypeptide" means a polypeptide chain having one or more glycosyl moieties attached thereto. No distinction is made herein to differentiate small glycopolypeptides from large glycopolypeptides or glycoproteins.
[0056] As used herein, the term "antibody" means a naturally produced antibody, recombinantly produced antibody, monoclonal antibody, or polyclonal antibody, as well as an antibody fragment such as an Fab fragment, F(ab')2 fragment, Fv fragment, or single-chain Fv fragment (scFv) that retains the relevant binding specificity of the native antibody. The term antibody is used to include antibodies of all immunoglobulin classes, such as IgM, IgG, IgD, IgE, IgA, and their subclasses.
[0057] As used herein, the term "predominant" indicates greater than 50%.
[0058] As used herein, the terms "increase" and "decrease" mean, respectively, to cause an increase or decrease of at least 5%, as determined by a method and sample size that achieves statistical significance (i.e., p < 0.01).
[0059] As used herein, the term "statistically significant" means having a probability of less than 1% under the relevant null hypothesis (i.e., p < 0.01).
[0060] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or." [0061] As used herein and in the appended claims, the use of singular forms of words, and the use of the singular articles "a," "an" and "the," are intended to include and not exclude the use of a plurality of the referenced term unless the context clearly dictates otherwise.
[0062] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, . . . , 0.9, 0.99, 0.999, or any other real values > 0 and≤ 2, if the variable is inherently continuous.
[0063] General Considerations
[0064] The present invention depends, in part, upon the discovery that the glycoslyation pathways of ciliates can be genetically modified to produce glycoproteins in which the glycans have a predominant core N-glycan structure which includes a
Man3GlcNAc2 N-linkage. In some embodiments, the mature glycoproteins comprise predominantly Man3GlcNAc2 N-linked glycans. In other embodiments, a Man3GlcNAc2 glycan is produced as an intermediate that is subsequently modified by additional endogenous or heterologous glycosylation enzymes, resulting in a processed glycan which retains the Man3GlcNAc2 core N-glycan.
[0065] Recombinant expression systems suitable for the production of clinically and economically important proteins using the methods described herein, include, but are not limited to, recombinant protein expression in Tetrahymena thermophila,
Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi,
Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis,
Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes octocarinatus, Euplotes vannus, Monoeuplotes crassus, Blepharisma japonicus. [0066] N-linked Glycan Biosynthesis in Yeast and Mammals
[0067] N-linked glycan biosynthesis occurs in a step-wise pathway in the endoplasmic reticulum and Golgi apparatus in a process that is well-conserved among many eukaryotes (for a review, see Chen et al. (2005), "Mammalian Glycosylation: An overview of carbohydrate biosynthesis," in Yarema, ed., Handbook of Carbohydrate Engineering, Boca Raton (FL), Dekkar CRC Press). In yeast and mammalian cells, assembly of the glycan precursor is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) membrane on a dolichol pyrophosphate (Dol-PP) isoprenoid lipid carrier by the addition of two GlcNAc and five Man residues (Figure 1). The Man5GlcNAc2-Dol-PP oligosaccharide is then flipped to the luminal face of the ER. In yeast and mammals, an additional four mannoses and three glucoses are added to the precursor to yield a Glc3Man9GlcNAc2-Dol-PP oligosaccharide (the initial core N-glycan) that is transferred en bloc to asparagine residues of certain proteins (Figure 1). Following co-translational transfer of the initial core N-glycan to the protein, the glucose residues are trimmed in a process that ensures correct folding. At that time, an a- 1,2- linked mannose is removed by an a-l,2-mannosidase to generate a MangGlcNAc2 processed core N-glycan that is present on glycoproteins as they exit the ER for later secretory compartments.
[0068] After exit of a glycoprotein from the ER, the glycans may be further modified by processing enzymes that add and/or remove sugars from the glycan structure. The nature and extent of these glycan remodeling reactions can differ. In yeast the addition in the Golgi of a single a-l,6-linked mannose to the a- 1,3 branch of the core glycan by the mannosyltransferase Ochl initiates synthesis of outer chain mannan, a structure that can decorate the glycan with well over 100 additional mannoses (Figure 2). Mannose addition is achieved through the action of a number of different mannosyltransferases (e.g., Mnnl, Mnn2, Mnn5, Mnn9, MnnlO, Mnnl 1, Anpl, Vanl, Hocl) and the resulting hyper- mannosylated glycans tend to be largely heterogeneous in nature (Jigami (2008), Biosci. Biotechnol. Biochem. 72:637-648).
[0069] The non-human nature of yeast N-glycans, and particularly the antigenic potential of hypermannosylated side chains and the associated phosphorylated terminal mannose residues, precludes the therapeutic use of recombinant proteins that are decorated with these N-linked glycans. [0070] In mammalian cells, N-glycans are processed to generate either hybrid or complex-type glycans (Figure 3). In both cases, the cis-Golgi mannosidase I first removes a- 1,2-linked mannoses to generate a MansGlcNAc2 oligosaccharide. A hybrid glycan is formed in the medial-Golgi by addition of a single a-l,2-linked GlcNAc to the a-l,3-linked mannose by GlcNAc transferase I. The remaining two outer mannose residues (a-1,3 and a-1,6) are subsequently removed through the action of mannosidase II to generate a
GlcNAciMan3GlcNAc2 oligosaccharide. A second a- 1,2-linked GlcNAc residue is then added by GlcNAc transferase II to generate a GlcNAc2Man3GlcNAc2 complex-type glycan. This is followed by the addition of two β-1,4 galactose residues by a galactose transferase and finally by the addition of terminal sialic acids by various sialyl transferases. It is important to note that complex glycan structures may display significant diversity, including tri and tetra-antennary structures that may or may not contain additional side-branching moieties such as a a-1,6 fucose (added in the medial Golgi by fucosyl transferase) or bisecting β-1,4 GlcNAc. The typical processed core N-glycan, however, is Man3GlcNAc2.
[0071] The composition and uniformity of N-linked glycosylation can be important for the production of therapeutic recombinant proteins. However, glycan uniformity can be difficult to control in mammalian heterologous protein expression systems such as CHO cells. Most glycoprotein therapeutics that are currently approved for human use contain a mixture of several different N-glycan structures.
[0072] Recently, systematic engineering of yeast N-glycosylation pathways (termed "glycoengineering") has resulted in the expression of glycoproteins bearing humanlike glycosylation pathways. Such methods have been used to produce a protein bearing a fully sialylated N-glycan with the structure NANA2Gal2GlcNAc2Man3GlcNAc2 in a yeast strain deficient in Ochl, to eliminate the outer chain mannan on yeast N-glycans (Gerngross (2004), Nat. Biotechnol. 22: 1409-1414; Li et al. (2006) Nat. Biotechnol. 24:210-215;
Hamilton et al. (2006) Science 313: 1441-1443). A Man5GlcNAc2 glycan that is identical to the mammalian core oligosaccharide produced in the cis-Golgi was produced by the introduction of an ER targeted a-l,2-mannosidase. The maturation of this glycan to a complex-type glycan was carried out in a manner analogous to mammalian N-glycan processing by the heterologous expression of various mammalian mannosidases, hexose transferases, nucleotide sugar transporters and CMP-sialic acid biosynthetic proteins (Figure 3). [0073] N-linked Glycan Biosynthesis in Ciliates
[0074] Ciliates are complex unicellular eukaryotes. For example, annotation of the fully sequenced genome and EST analysis of the ciliate Tetrahymena thermophila indicates that this organism has approximately 24,000 genes (Eisen et al. (2006), PLoS Biol. 4: 1620-1642; Coyne et al. (2008), BMC Genomics 9:562). Large extended gene families of ABC transporters, GPI-anchored proteins and voltage-gated ion channels are indicative of the complexity of ciliates. Indeed the presence in T. thermophila of over 400 potassium ion channels rivals that of human cells. Despite their apparent complexity, ciliates in general, and Tetrahymena in particular, still enjoy the benefits of a microbial expression platform, including fast generation times (1.5-3h), scalability, and growth on a number of inexpensive complex and defined media.
[0075] Figure 4 is a schematic representation of the putative major pathway of N- glycan synthesis in ciliates, and illustrates the differences from the putative major pathway in yeast and mammalian cells depicted in Figure 1. Specifically, comparing Figures 1 and 4, although ciliates contain the enzymes necessary for steps 1 through 8 shown in both figures, they lack the enzymes necessary for steps 9 through 12 of Figure 1. Thus, like yeast and mammalian cells, the primary glycosylation pathway leads to the production of a
Man5GlcNAc2 Dol-PP oligosaccharide intermediate. However, because they lack the Alg3, Algl2, Alg9 mannosyltransferases, the initial core N-glycan produced by ciliates is
Glc3Man5GlcNAc2 (Figure 4), rather than the Glc3Man9GlcNAc2 initial core N-glycan produced by yeast and mammals (Figure 1). The structures of these alternative core N- glycans are depicted in Figure 5.
[0076] In ciliates, as in yeast and mammalian cells, after the glycan Dol-PP oligosaccharide is transferred to a protein by an oligosaccharyltransferase complex (OST), the three Glc residues are removed by ER resident a glucosidases I and II.
[0077] In yeast and mammals, this results predominantly in Man9GlcNAc2 N-glycans. In ciliates, however, this results predominantly in MansGlcNAc2 N-glycans. In addition, in yeast and mammals, but not in ciliates, an a-l,2-mannosidase typically removes an additional Man residue, to produce Man8GlcNAc2 N-glycans.
[0078] Furthermore, because ciliates lack a classic Golgi apparatus or Golgi bodies (see, e.g., Elliott and Zieg (1968), J. Cell Biol. 36(2):391-398) and, therefore, Golgi- associated enzymes, they do not produce the elaborate complex and hybrid structures generated in the Golgi of yeast or mammalian cells (Figures 2 and 3). Additional processing can include removal of up to three mannose residues by one or more yet unidentified enzymes with a-l,2-mannosidase, a-l,3-mannosidase and/or a-l,6-mannosidase activity. Such mannose trimming may occur intracellulary in the compartment of the secretory pathway and/or in the extracellular media.
[0079] This is confirmed by structural analysis of glycans released from native and recombinant proteins produced in ciliates, which bear a mixture of Man2GlcNAc2, Man3GlcNAc2, Man4GlcNAc2 and Man5GlcNAc2 N-glycans (Taniguchi et al. (1985), J. Biol. Chem. 260: 13941-13946; Weide et al. (2006), BMC Biotechnol. 6: 19; Becker and Rusing (2003), J. Eukaryot. Microbiol. 50:235-239). While the relative ratio of each glycoform may differ depending on the protein(s) analyzed, the major (but not predominant) glycoform present on most proteins expressed in ciliates, whether native or recombinant, is
Man3GlcNAc2 (Taniguchi et al. (1985), J. Biol. Chem. 260: 13941-13946; Weide et al.
(2006), BMC Biotechnol. 6: 19).
[0080] The putative major glycosylation pathway in ciliates described above is also consistent with bioinformatic analysis. For example, the sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia reveals homologs of all the yeast and mammalian genes required to produce N-glycans as shown in steps 1 through 16 of Figure 1 with the notable absence of the genes encoding the Alg3, Alg9 and Algl2- mannosyltransferases required for steps 9 through 12. In addition, the ciliates lack homologs to many other yeast and mammalian genes involved in glycosylation (Table 1).
TABLE 1. Bioinformatic identification of N-glycosylation proteins in sequenced ciliate genomes1
Protein Enzyme Yeast Human T. thermophila P. tetraurelia2
N-glycan precursor synthesis enzymes
UDP-N-acetyl-
Alg7p glucosamine-l-P NP_009802 NP_001373 XP_001007837 XP_001425486 transferase
catalytic subunit of
Algl3p UDP-GlcNAc NP_011468 NP 060936 XP_001033121 XP_001439007 transferase
second subunit of UDP- XP 001445130/
Algl4p NP 009626 NP_659425 XP_001019482
GlcNAc transferase XP 001444700 chitobiosyldiphospho-
Alglp NP 009668 NP_061982 XP_001018043 XP_001424991 dolichol b-mannosyl transferase
a- 1 ,3/1 ,6-mannosyl
Alg2p NP_011450 NP_149078 XP_001017562 XP_001450661 transferase
a-l,2-mannosyl
Algl lp NP_014350 NP_001004127 XP 001019991 XP_001428423 transferase
a-l,3-mannosyl
Alg3p NP_009471 NP_005778 absent absent
transferase
a-l,2-mannosyl
Alg9p NP_014180 NP_079016 absent absent
transferase
a-l,6-mannosyl
Algl2p NP_014427 NP_077010 absent absent
transferase
a-l,3-glucosyl
Alg6p NP_014644 NP_037471 XP_001022240 XP_001448391 transferase
a-l,3-glucosyl
Alg8p NP_014710 NP_076984 XP_001016294 XP_001428028 transferase
a-l,2-glucosyl
AlglOp NP_011251 NP_116223 XP_001021336 XP_001447557 transferase
N-glycan transfer to proteins (oligosaccharyltransferase complex)3
oligosaccharyl
XP 001423896/
Ostlp transferase (OST) NP_012532 NP_002941 XP_001014582
XP_001436757 subunit
oligosaccharyl
XP 001458694/
Ost2p transferase (OST) NP_014746 NP_001335 XP_001020847
XP_001461596 subunit
oligosaccharyl
Swplp transferase (OST) NP_013869 absent absent absent
subunit
oligosaccharyl
XP 001455366/
Wbplp transferase (OST) NP_010914 NP_005207 XP_001013892
XP_001425674 subunit
oligosaccharyl
NP 849193/
Stt3p transferase (OST) NP_011493 XP_001016052 XP_001439477
NP 689926
subunit
ER glycan processing
Mnslp a- 1 ,2-mannosidase NP O 12665 NP_057303 absent absent
Rot2p glucosidase II NP_009788 NP_938148 XP_001024992 XP_001454030
Cwh41p glucosidase I NP_011488 NP 006293 XP_001032354 XP_001451144
Mammalian Golgi complex-type N-glycan maturation enzymes
MANIA
Golgi a-mannosidase I absent NP_005898 absent absent 1
MAN2A
Golgi α-mannosidase II absent NP_002363 XP_001014314 XP_001443250 1
mannosyl (a-1,3-)- glycoprotein
MGAT1 β-1,2-Ν absent NP_001108089 absent absent
acetylglucosaminyl- transferase
mannosyl (a-1,6-)- glycoprotein
MGAT2 β-1,2-Ν- absent NP 002399 absent absent
acetylglucosaminyl- transferase mannosyl (β-1,4-)- glycoprotein
MGAT3 β-1,4- absent NP_001091740 absent absent
Nacetylglucosaminyl- transferase
mannosyl (a-1,3-)- glycoprotein
MGAT4 β-1,4-Ν- absent NP 036346 absent absent acetylglucosaminyl- transferase
mannosyl (a-1,6-)- glycoprotein
MGAT5 β-Ι,ό-Ν-acetyl- absent NP_002401 absent Absent glucosaminyl- transferase
a-l,6-fucosyl
FUT8 absent NP_835368 absent Absent transferase
B4GAL- β-1,4- galactosyl
absent NP_001488 absent Absent Tl transferase 1
ST3GAL β-galactoside a-2,3- absent NP 006091 absent Absent 6 sialyltransferase 6
ST6GAL β-galactosamide a-2,6- absent NP_003023 absent Absent 1 sialyltranferase 1
ST6GAL β-galactosamide a-2,6- absent NP_115917 absent Absent
2 sialyltranferase 2
a-N-acetyl-neuraminide
ST8SIA2 absent NP_006002 absent Absent a-2,8-sialyltransferase 2
a-N-acetyl-neuraminide
ST8SIA4 absent NP 005659 absent Absent a-2,8-sialyltransferase 4
a-N-acetyl-neuraminide
ST8SIA3 absent NP 056963 absent Absent a-2,8-sialyltransferase 3
Yeast Golgi N-glycan outer chain mannan synthesis
a- 1,6 mannosyl
Ochlp NP_011477 absent absent Absent transferase
a- 1,6 mannosyl
An l NP_010878 absent absent Absent transferase
a- 1,6 mannosyl
Hoclp NP O 12609 absent absent Absent transferase
Subunit of a Golgi
Mnn9p mannosyltransferase NP_015275 absent absent Absent complex
Subunit of a Golgi
MnnlOp mannosyltransferase NP_010531 absent absent Absent complex
Subunit of a Golgi
Mnnl lp mannosyltransferase NP_012352 absent absent Absent complex
Component of the
Vanlp NP_013592 absent absent Absent mannan polymerase I
a-l,2-mannosyl
Mnn2p NP_009571 absent absent Absent transferase
a-l,2-mannosyl
Mnn5p NP_012349 absent absent Absent transferase
a- 1,3 -mannosyl
Mnnlp NP 010916 absent absent Absent transferase
Ktr6p mannosylphosphate NP_015272 absent absent Absent transferase
'Proteins were identified in sequenced geneomes using the BlastP algorithm and the S. cerevisiae (yeast) or human protein sequences as queries. Probable proteins are indicated by the presence of a GenBank accession number.
2In certain cases, two high scoring putative proteins were identified and both accession numbers are shown.
3The OST complex in yeast contains 9 subunits (Ostlp-Ost6p, Stt3p, Swplp and Wbplp) with only 5 being essential for viability (Ostlp, Ost2p, Stt3p, Swplp and Wbplp). Other non-essential subunits are typically short hydrophobic peptides and are therefore difficult to identify in the genomes of distantly related organisms using Blast homology analysis. Therefore, only the essential components of OST are shown in this table to illustrate that some related form of OST is present in ciliates.
[0081] The presence of glyco forms containing different mannose content in ciliate N-glycans shows that, like mammalian and yeast cells, there is some degree of variation in the processing of the Glc3Man5GlcNAc2 initial core glycans after they are attached to proteins. Unlike yeast and mammalian cells, however, where processing of the initial core N- glycan Glc3Man9GlcNAc2 involves the orchestrated removal and then addition of
carbohydrate moieties, processing of the glycan precursor in ciliates involves only removal of the three Glc residues followed by mannose trimming through the action of a protein having a-l,2-mannosidase activity that generates Man4GlcNAc2 and Man3GlcNAc2 glycans, and either an a- 1,3 or an a-l,6-mannosidase that generates Man2GlcNAc2 glycans. At present it is unknown in which cellular compartment this processing of the ciliate glycans is occurring. One possibility is that this activity occurs in late stage secretory vesicles following transit of glycoproteins from the ER. The fact that secreted a- 1,2, a- 1,3 and a-l,6-mannosidase activities have been observed in media from Tetrahymena cultures (Banno et al. (1987), Exp. Cell Res. 170:259-268; Blum (1976), J. Cell Physiol. 89:457-72) raises the possibility that mannose trimming events may occur with exposure to the extracellular medium.
[0082] Genetically Modified Ciliates Expressing a-l,2-mannosidase
[0083] In one aspect, the present invention provides genetically modified ciliates which produce glycoproteins with predominantly or substantially uniform Man3GlcNAc2 core N-glycans. In some embodiments, this is achieved through the production of genetically modified ciliates that express an exogenous a-l,2-mannosidase which significantly increases the percentage of Man3GlcNAc2 core N-glycans relative to Man4GlcNAc2 and MansGlcNAc2 core N-glycans. [0084] Depending upon the level of expression and the activity of the a- 1 ,2- mannosidase in the genetically-modified ciliates, the invention can achieve different percentages of Man3GlcNAc2 core N-glycans on glycoproteins produced by the ciliates. In some embodiments, the glycoproteins produced by the genetically-modified ciliates will have predominantly Man3GlcNAc2 core N-glycans. In embodiments with higher levels of a- 1,2- mannosidase expression or activity, the percentage of total N-glycans having a Man3GlcNAc2 core N-glycan structure can range from 50%- 100%, including all values in between. In certain preferred embodiments, the percentage of Man3GlcNAc2 core N-glycans can be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[0085] The a-1 ,2-mannosidase, or protein having a-1 ,2-mannosidase activity, used in conjunction with the methods described herein, can be from any source. In one embodiment, as shown in the Examples below, an expression cassette can be designed to express an a-l,2-mannosidase (GenBank Gene ID, 3408991) from the protist Entamoeba histolytica.
[0086] Additional or alternative a-l,2-mannosidases suitable for use in the methods described herein include, but are not limited to: Aspergillus saitoi a- 1,2- mannosidase (Ichishima et al. (1999), Biochem. J. 339(Pt 3):589-597), Trichoderma reesei a-l,2-mannosidase (Maras et al. (2000), J. Biotechnol. 77(2-3):255-263), Penicillium citrinum a-l,2-mannosidase (Yoshida et al. (1993), Biochem. J. 290(Pt 2):349-354),
Aspergillus nidulans a-l,2-mannosidase (Eades and Hintz (2000), Gene 255(l):25-34), Homo sapiens a-l,2-mannosidase I A, Homo sapiens IB a- 1,2- mannosidase, Lepidopteran insect Type I a-l,2-mannosidase (Ren et al. (1995), Biochem. 34(8):2489-2495), Homo sapiens aD mannosidase (Chandrasekaran et al. (1984), Cancer Res. 44(9):4059-68), Xanthomonas a-l,2-mannosidase, Mouse IB a-l,2-mannosidase (Schneikert and Herscovics (1994), Glycobiology 4(4):445-50) and Bacillus sp. a-l,2-mannosidase (Maruyama et al. (1994), Carbohydrate Res. 251 :89-98).
[0087] In accordance with the present invention, an a-l,2-mannosidase is expressed in a ciliate and targeted to a site in the secretory pathway where at least one
Man3GlcNAc2 N-glycan structure, at least one Man4GlcNAc2 N-glycan structure (a substrate of a-l,2-mannosidase), or at least one Man5GlcNAc2 N-glycan structure (a substrate of a- 1,2- mannosidase) is present. In some embodiments, the a-l,2-mannosidase is targeted to the ER.
In other embodiments, the a-l,2-mannosidase is targeted to cortical granules or mucocysts. [0088] In order to achieve accumulation of a-l,2-mannosidase activity in the ER or an ER-derived storage or secretory vesicle, a polypeptide having a-l,2-mannosidase activity can be linked to an ER-targeting signal that causes the polypeptide to be targeted to and/or retained in the ER or ER-derived storage or secretory vesicle. In some embodiments, the a-l,2-mannosidase is localized to the ciliate ER through targeting sequences which are native to the mannosidase-coding sequences. In other embodiments, the ER targeting sequences are heterologous to the mannosidase-coding sequences.
[0089] An "ER targeting sequence" refers to a peptide sequence which directs a protein having such a peptide sequence to be transported to and/or retained in the ER or an ER-derived storage or secretory vesicle. Such ER targeting sequences are often found in proteins that reside and function in the ER, and multiple such ER targeting signals suitable for use with the methods described herein can readily be identified by one skilled in the art. The ER targeting sequences can be N-terminal or C-terminal. In certain embodiments, a targeting sequence that is capable of retaining a polypeptide in the ER contains a C-terminal ER-retention motif. Examples of such C-terminal ER-retention motifs include KDEL, HDEL, DDEL, ADEL and SDEL sequences, as well as those ER-retention sequences disclosed in Xie et al. (2007), Eukaryotic Cell 6(3):388-397 and Andres et al. (1991), J. Biol. Chem. 266(22): 14277-14282.
[0090] Other targeting sequences are well known and described in the scientific literature and public databases (see http://www.ciliate.org/genetics.shtml). Such targeting sequences include, but are not limited to, the ER retention sequences from the Tetrahymena GPI transamidase complex. The GPI transamidase complex is involved in the transfer of lipid-linked GPI precursor molecules to target proteins in the ER. For example, the C- terminal portion (54 amino acids) of the PigT component of the GPI transamidase complex (which includes a transmembrane domain), can be fused to a recombinant protein to maintain ER localization. Similarly, any Tetrahymena ER resident protein, or portion thereof, having ER retention activity can be used as a fusion partner to promote ER localization of the fusion partner.
[0091] In certain cases, an a-l,2-mannosidase from a non-ciliate species may not have an ER targeting sequence, or will not have an ER targeting sequence which functions well in a ciliate. In such cases, one skilled in the art will readily be able to modify such an a-
1 ,2-mannosidase by adding an appropriate ER targeting sequence to the a-l,2-mannosidase by standard protein engineering techniques. In cases where the a-l,2-mannosidase contains one or more non-ER targeting signals (e.g., a signal that may direct the a-l,2-mannosidase to another subcellular compartment or organelle), one skilled in the art will readily be able to modify the protein to remove any non-ER targeting sequences and to add an appropriate ER targeting sequence (e.g., as disclosed in Xie et al. (2007), Eukaryotic Cell 6(3):388-397).
[0092] Instead of, or in addition to, an ER targeting sequence, in certain embodiments, the a-l,2-mannosidases can further comprise targeting sequences that direct localization of the a-l,2-mannosidase to one or more other subcellular compartments or organelles. For example, in certain other embodiments, the a-l,2-mannosidase can be localized to a ciliate's mucocysts through fusion of the a-l,2-mannosidase to a polypeptide having a mucocyst retention or targeting signal. Exemplary polypeptides having mucocyst targeting sequences suitable for use with the methods described herein include both full length granule lattice proteins (e.g., Girl), the "pre-pro" domains of granule lattice proteins, and Igr proteins.
[0093] Ciliates that include mucocysts useful in the invention include
Tetrahymena species such as Tetrahymena thermophila and Tetrahymena pyriformis. Both Tetrahymena thermophila and Tetrahymena pyriformis produce mucocysts, and both can produce a heterologous protein which will be secreted within the proteinaceous gel which results from mucocyst discharge. Paramecium has dense core granules but does not secrete a proteinaceous gel. In other embodiments, the a-l,2-mannosidase can be localized to a ciliate constitutive secretory pathway either through localization sequences native to the a- 1,2- mannosidase or through fusion of the mannosidase to a polypeptide having a constitutive secretory pathway targeting signal. An exemplary polypeptide having a secretory pathway targeting signal is the signal peptide from the immobilization antigen variant B protein of Ichthyophthirius multifiliis (MKFNILIILIISLFINELRA (SEQ ID NO: 1)). Other polypeptide sequences having secretory pathway targeting activity can be identified by those skilled in the art using standard algorithms (e.g. , SignalP) suitable for detecting the presence and location of signal peptides in amino acid sequences. Exemplary Tetrahymena polypeptides having secretory pathway targeting activity include, but are not limited to those polypeptides listed in Table 2.
Table 2. Tetrahymena polypeptides having secretory pathway targeting activity Tetrahymena Genome Database Sequence
Protein Identification Number
61740 MNKFSLVLCLSLVLISLRAES (SEQ ID NO: 2)
79650 MSKLLIAVLLCLAIITPTVLC (SEQ ID NO: 3)
590090 MRS STLSILLLCILGASIA (SEQ ID NO: 4)
442170 MQINRVIFWSALIAVSLLTGFA (SEQ ID NO: 5)
606960 MQNKTIIICLIISQLLVS VF S S AGGQA (SEQ ID NO: 6)
418110 MNTKLLIALPILALLSIGAVFL (SEQ ID NO: 7)
683010 MNKTLILALVGVLALTATTLVA (SEQ ID NO: 8)
66890 MNKLVLIALVTLFAGVMA (SEQ ID NO: 9)
1068140 MQKSIIIAAILLVGLASA (SEQ ID NO: 10)
220690 MNKTSIILIASILSAALCGA (SEQ ID NO: 11)
321640 MNKVALIASFLAGLSILSIS (SEQ ID NO: 12)
227830 MKTQFVFLFTLLLLNALC (SEQ ID NO: 13)
411610 MSKLQFVLIAALLLVAVSA (SEQ ID NO: 14)
149680 MNQKQLF ALLVIVFIQIS ST SC (SEQ ID NO: 15)
196530 MKKIALLSVCAFILLISFANC (SEQ ID NO: 16)
701150 MAALKILLVAILLCSGCLS (SEQ ID NO: 17)
[0094] In other embodiments, the a-l,2-mannosidase does not contain any native or heterologous targeting or localization sequences.
[0095] The a-1 ,2-mannosidase for use in the present invention can be further modified by, for example, insertion of an epitope tag to which antibodies are available {e.g. , Myc, HA, FLAG and His6 tags) by methods well-known in the art. An epitope-tagged a- 1,2- mannosidase can be conveniently purified, or monitored for both expression and intracellular localization.
[0096] In one embodiment, the a-l,2-mannosidase is expressed from an expression cassette suitable for expressing the a-l,2-mannosidase in the ciliate. Such expression cassettes can comprise regulatory elements, such as a promoter and/or a terminator, as well as optional sequences for positive or negative selection, such as sequences conferring resistance or susceptibility. Such expression cassettes can be maintained in either the ciliate macronucleus or in the micronucleus.
[0097] In another embodiment, the expression cassette encoding a protein having a-l,2-mannosidase activity can further comprise a transgene for the expression of a recombinant polypeptide of interest (e.g., a therapeutic polypeptide). Polypeptides encoded by transgenes suitable for expression by the genetically modified ciliates described herein are not limited to any particular protein and can include any polypeptide which it is desirable to produce with predominantly or substantially uniform Man3GlcNAc2 core N-glycans.
[0098] Any method known in the art can be used to select for the genetically modified ciliates described herein. Suitable methods include, but are not limited to, selection for resistance to an antibiotic. In certain embodiments, phenotypic assortment can also be used to select for one or more genetically modified ciliates suitable for use with the methods described herein.
[0099] The genetically modified ciliates having significantly increased a- 1,2- mannosidase activity can also have significantly reduced a-l,2-mannosyltransferase activity, as described below.
[00100] Genetically Modified Ciliates Having Reduced a-l,2-Mannosyltransferase Activity
[00101] As shown in Figures 1 and 4, Algl 1 is an a-l,2-mannosyltransferase that sequentially adds two a-l,2-linked mannose residues to the Man3GlcNAc2 Dol-PP glycan precursor on the cytoplasmic face of the ER to produce a MansGlcNAc2 Dol-PP glycan precursor, which is then translocated to the luminal face of the ER. In ciliates, as shown in Figure 4, no further mannose residues are added before the enzymes Alg6, Alg8 and AlglO sequentially add three glucose residues to the terminal a-l,2-linked mannose residue prior to transfer of the initial core N-glycan to the nascent glycoprotein. By reducing or eliminating expression of Algl 1, therefore, genetically-modified ciliates can be engineered which will produce a predominantly or substantially uniform Glc3Man3GlcNAc2 initial core N-glycan.
[00102] Accordingly, in one aspect, the invention provides a glycoprotein expression system wherein genetically engineered ciliates, in which at least one Algl 1 gene locus has been deleted, disrupted or modified to have significantly reduced expression, are used to produce the glycoproteins containing predominantly Man3GlcNAc2 core N-glycans. In another aspect, the invention provides a glycoprotein expression system wherein genetically engineered ciliates having substantially reduced a-l,2-mannosyltransferase activity, as compared to a ciliate that has not been genetically modified, are used to produce the glycoproteins containing predominantly or substantially uniform Man3GlcNAc2 core N- glycans.
[00103] In some embodiments, the reduction or loss of Algl 1 function is due to a genetic modification in an Algl 1 gene locus, including, but not limited to, one or more deletions, mutations or substitutions in a micronuclear Algl 1 gene. In other embodiments, the reduction or loss of Algl 1 function is due to a genetic modification in the Algl 1 gene locus, including, but not limited to, one or more deletions, mutations or substitutions in a macronuclear Algl 1 gene. Genetic modifications suitable for use with the methods described herein include any genetic modifications that inactivate or reduce expression of Algl 1, inactivate or reduce 1 ,2-mannosyltransferase activity of a protein encoded by Algl 1, and/or mislocalize a 1,2-mannosyltransferase protein encoded by Algl 1.
[00104] In some embodiments, one or more inhibitory R As (iR As) can be used to silence a- 1,2-mannosyltransferase expression or a- 1,2-mannosyltransferase activity by RNA interference (RNAi) alone or in combination with any other methods described herein. RNAi is a process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in cells (Hutvagner and Zamore, 2002, Curr. Opin. Genet. Dev. 12:225-232; Sharp, 2001, Genes Dev. 15:485-490).
[00105] "Silencing" a target gene means the process whereby a cell containing and/or secreting a certain product of a target gene when not in contact with the agent, will contain and/or secret at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.
[00106] An "iRNA agent" as used herein, is an RNA agent, which can down- regulate the expression of a target gene, e.g., a a- 1,2-mannosyltransferase gene. An iRNA agent {e.g., a double stranded iRNA agent or an small inhibitory RNA agent) may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mR A sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms.
[00107] RNAi can be used in ciliates (e.g., Tetrahymena spp.) to silence gene expression (Howard-Till and Yao (2006) Mol. Cel. Biol. 26, 8731-8742). In some embodiments, expression of a target gene {e.g., a-l,2-mannosyltransferase gene) can be silenced with RNAi technology by targeting nucleotide sequences complementary to any region of the sequence encoding the target protein {e.g., a-l,2-mannosyltransferase) to form structures that prevent transcription of the gene in target cells. For example, a 400 to 500 base pair length of a target gene can cloned in an inverted orientation around a 90-bp linker containing a 50-bp intron from an unrelated gene. This hairpin construct can then cloned into an rDNA vector under the control of a promoter, for example an inducible MTT promoter. In some embodiments, the iRNA agents can be used to silence expression of a- 1,2- mannosyltransferase can comprise about 16 to about 500 nucleotides in each strand, wherein one of the strands contains a region having substantially complementary to, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) complementary {e.g., having zero or one or more mismatched nucleotides) to a region {e.g., a transcribed region or a regulatory region) in a- 1 ,2-mannosyltransferase mRNA.
[00108] iRNA agents, as described herein, can mediate silencing of a gene, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a gene is also referred to as a target gene. In certain embodiments, the RNA to be silenced is aciliate a-l,2-mannosyltransferase gene. In certain embodiments, the iRNA agent can be a double stranded (ds) iRNA agent. A "ds iRNA agent", as used herein, is an iRNA agent which includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure. A "strand" herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g., by a linker, e.g., a polyethyleneglycol linker, to form but one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA {e.g., a-l,2-mannosyltransferase mRNA). Such strand is termed the "antisense strand". A second strand comprised in the dsRNA agent which comprises a region complementary to the antisense strand is termed the "sense strand." However, a ds iRNA agent can also be formed from a single RNA molecule which is, at least partly; self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.
[00109] The iRNA agents suitable for use with the methods described herein can be delivered to a cell by methods known in the art and as described herein. For example, in some embodiments, the iRNA agent can be expressed from an expression vector or the iRNA agent can be delivered directly into a cell by transformation according to any of the methods described herein. The iRNA agents can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from an engineered RNA precursor. The iRNA agents may be designed using methods known in the art (e.g., "The siRNA User Guide," available at Rockefeller edu/labheads/tuschl/siRNA).
[00110] The effectiveness of an iRNA agent in silencing expression of a-1,2- mannosyltransferase can be assessed by any of a number of assays following introduction of the into a cell. These assays include, but are not limited to, Western blot analysis using antibodies that recognize the targeted a-l,2-mannosyltransferase gene product following sufficient time for turnover of the endogenous pool after new protein synthesis is repressed, and Northern blot analysis to determine the level of existing target a-l,2-mannosyltransferase mRNA. Negative control iRNAs can be designed to have generally have the same nucleotide composition as the selected iRNA agent, but without significant sequence complementarity to the targeted genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected iRNA agent; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control iRNAs can be designed by introducing one or more base mismatches into the sequence. Such negative controls are used to, e.g., confirm the specificity of a test iRNA agent.
[00111] An iRNA agent useful for silencing expression of a- 1,2- mannosyltransferase can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al. (1996), Bioorganic & Medicinal Chemistry 4: 5-23). [00112] In another embodiment, the genetically modified ciliates can further comprise a transgene for the expression of a recombinant polypeptide of interest (e.g. , a therapeutic polypeptide). Polypeptides encoded by transgenes suitable for expression by the genetically modified ciliates described herein are not limited to any particular protein and can include any polypeptide which it is desirable to produce with predominantly or substantially uniform Man3GlcNAc2 core N-glycans.
[00113] Any method known in the art can be used to select for the genetically modified ciliates described herein. Suitable methods include, but are not limited to, selection for resistance to an antibiotic. In certain embodiments, phenotypic assortment can also be used to select for one or more genetically modified ciliates suitable for use with the methods described herein (see http://www ciliate.org/genetics.shtml).
[00114] The genetically modified ciliates having significantly reduced a- 1 ,2- mannosyltransferase activity can also have significantly increased a-l ,2-mannosidase activity, as described above.
[00115] Generation of Genetically Modified Ciliates
[00116] The genetically-modified ciliates of the invention can be produced by any means of genetic modification known in the art. In some embodiments, the ciliates are transformed with an expression vector which expresses a heterologous protein at least transiently, and which may or may not include sequences necessary for autonomous replication. In other embodiments, the ciliates are transformed with integration vectors which can cause a gene encoding a heterologous protein to be integrated into either the micronuclear or macronuclear genome. Integration vectors may cause random integration of the desired heterologous sequences, or may promote homologous recombination whereby the
heterologous sequences are inserted at a specific desired location in the ciliate genome.
[00117] The vectors described herein can comprise regulatory elements which are suitable for promoting expression in a ciliate host, or regulatory elements can be supplied by an endogenous gene into which a heterologous coding region integrates. Suitable regulatory regions include, but are not limited to, promoters, enhancers, termination sequences, polyadenylation signals, signal polypeptides and proprotein domains involved in the expression and secretion of proteins. For example, such regulatory elements can provide efficient heterologous expression of heterologous proteins in ciliates under control of promoters and/or terminators which are derived from ciliate genes. Such vectors can comprise naturally occurring promoters and/or terminators from proteins secreted at a high level in ciliates. In some embodiments, the promoters and/or terminators can be selected from proteins secreted at a high level independent of the cell-cycle (U.S. Patent Publication No. 2006/0127973; PCT Intl. Pub. WO2003/078566). Inducible promoters from ciliate genes have also been described that allow expression of heterologous genes. For example, heat- inducible promoters of the heat shock protein family of ciliates are also suitable for use with the methods described herein. Suitable heat shock promoters from Tetrahymena spp. are known in the art (see, .e.g., PCT Intl. Pub. WO2007/006812).
[00118] Such vectors can further comprise a 5' regulatory region from an endogenous ciliate gene containing a promoter region operably joined to the heterologous coding region and/or a 3' regulatory region from the same or a different ciliate gene. Suitable regulatory regions from ciliate genes are well known in the art.
[00119] Vectors suitable for use with the methods described herein include, but are not limited to: the pXS76 shuttle vector (which can be used for insertion of transgenes downstream of a cadmium-inducible promoter from the MTTl metallothionein gene), rDNA vectors (Tondravi et al. (1986), Proc. Natl. Acad. Sci USA 83:4396; Yu et al. (1989), Proc. Natl. Acad. Sci USA 86: 8487-8491), and high copy number ribosomal DNA vectors (such as pD5H8). For example, rDNA-based vectors can be circular vectors containing 5' non- translated sequences comprising two or more ori sequences from a ciliate. One or more nucleic acid fragments containing heterologous coding regions {e.g., a selectable marker, a- 1 ,2-mannosidase gene, a-l,2-mannosyltransferase knock-out construct, or other transgene) can also be added to the vector using methods known to those skilled in the art. Such vectors can further comprise 5' untranslated regions of a ciliate gene and a 3' untranslated regions of a ciliate gene. These untranslated regions can be inserted upstream and downstream of the selectable marker and/or the transgene.
[00120] The nucleic acid sequences described herein can be cloned using standard cloning procedures in the art, as described by Sambrook et al, eds., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989). [00121] One skilled in the art will also appreciate that for a transgene to be expressed in a ciliate, such as Tetrahymena, additional factors may be considered to enhance protein expression, processing and/or trafficking in a ciliate. For example, it is possible to modify transgenes such that codon usages specific for a ciliate such as Tetrahymena is employed (Wuitschick and Karrer (1999), J. Eukaryot. Microbiol. 46(3):239-47).
[00122] Methods for creating mitotically stable ciliate transformants, for example, by integration of a heterologous gene by homologous DNA recombination, are known in the art. For example, methods for generating Tetrahymena spp. having targeted gene knockouts by homologous DNA recombination are described in Bruns & Cassidy-Hanley (1999), Methods Cell Biol. 62: 501-512; Hai et al. (1999), Methods Cell Biol. 62:514-531; Gaertig et al. (1999), Nature Biotech. 17:462-465; Cassidy-Hanley et al. (1997), Genetics 146: 135- 147).
[00123] The genetically modified ciliates described herein can be created using standard techniques known in the art. For example, ciliate cells can be transformed with a homologous recombination vector capable of replacing one or more a- 1,2- mannosyltransferase genes in the micronucleus with a nucleic acid sequence that reduces or abolishes activity or expression of the a-l,2-mannosyltransferase.
[00124] Expression vectors capable of homologous recombination with a highly expressed gene that is endogenous to the ciliate host, such as a β-tubulin 1 gene, are known in the art (see for example, U.S. Patent Application No. 2005/0106164). T. thermophila expresses two major β-tubulin genes, BTU1 and BTU2, which encode identical β-tubulin proteins (Gaertig et al. (1993), Cell Mot. Cytoskel. 25:245-253). Either of these two genes (but not both at once) can be disrupted without a detectable change in the cell phenotype.
[00125] Homologous recombination vectors useful for generating genetically modified ciliates having reduced or abolished a-l,2-mannosyltransferase expression or activity, include, but are not limited to knockout cassettes suitable for replacing the micronuclear DNA segment extending from a sequence between the regions flanking all or part of the micronuclear a-l,2-mannosyltransferase locus.
[00126] Such homologous recombination vectors can further comprise a selection marker such that cells having reduced or abolished a-l,2-mannosyltransferase expression or activity can be selected by growth in a biostatic or biocidal drug. Various methods for selecting cells that have undergone homologous recombination are well known in the art. The selection marker enables selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic or biocidal drugs. In addition, a heterologous gene that is to be expressed in the cell also may be included within the construct.
[00127] Any of the vectors described herein can be generated using molecular cloning techniques that are well-known in the art. Further, techniques for the procedures of molecular cloning can be found in Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Glover et al.
(1985), DNA Cloning: A Practical Approach, Volumes I and II, IRL Press, Washington, DC; Gait et al., (1985), Oligonucleotide Synthesis, IRL Press, Washington, DC; Hames and Higgins (1985), IRL Press, Washington, DC; Hames and Higgins (1984), Transcription And Translation, IRL Press, Washington, DC; Freshney et al. (1986), Animal Cell Culture, IRL Press, Washington, DC; Perbal (1986), Immobilized Cells And Enzymes, IRL Press,
Washington, DC; Perbal (1984), A Practical Guide To Molecular Cloning. John Wiley & Sons, Inc., New York; Ausubel et al. (2002), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
[00128] Vectors can be introduced into ciliates using established protocols or any method known to one skilled in the art. For example, transformation of ciliates can be achieved by microinjection (Tondravi and Yao (1986), Proc. Natl. Acad. Sci. USA 83:4369- 4373), electroporation (Gaertig and Gorovsky (1992), Proc. Natl. Acad. Sci. USA 89:9196- 9200), or biolistically (Cassidy-Hanley et al. (1997), Genetics 146: 135-147). Transformation of the either the somatic macronucleus or the generative micronucleus is also possible in ciliates which possess both, including Tetrahymena spp.
[00129] In some embodiments, ciliate cells can be transformed with a chimeric gene by particle bombardment (also known as biolistic transformation) (Cassidy-Hanley et al.
(1997), Genetics 146: 135-147). Particle bombardment transformation can be achieved by several ways. For example, inert or biologically active particles can be propelled at cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the chimeric gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Other variations of particle bombardment, now known or hereafter developed, can also be used.
[00130] Microcarrier bombardment can also be used to transform ciliate cells by means of DNA-loaded gold particles (US 6,087,124; EP 847 444; WO 1998/001572). In this approach, microcarrier bombardment with DNA-coated gold is used as a means of introducing heterologous genes into ciliates. In one embodiment, microcarrier bombardment can be used to transform ciliates and introduce genes into the (germline) micronucleus
[00131] Methods for selection of transformed cells harboring heterologous genes are known in the art. For example, the vector can further comprise a selectable cassette marker to permit selection for transformed cells (e.g., a neo 2 cassette) (Gaertig et al. (1994), Nucleic Acids Res. 22:5391-5398). Selection of transformants can be achieved by growing the cultured ciliates in a medium which allows only the transformants to survive. Suitable selection agents include antibiotics which will kill most all non-transformants but allow transformants (which also possess an antibiotic resistance gene) to survive. A number of antibiotic-resistance markers are known in the art (Debono and Gordee (1994) Annu. Rev. Microbiol. 48:471-497). Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention.
[00132] Methods of Producing Glycoproteins
[00133] In one aspect, the present invention provides glycoproteins having predominantly Man3GlcNAc2 core N-glycans. In these methods, a genetically-modified ciliate, as described herein, is used to express a heterologous glycoprotein. The glycoproteins are then isolated from the cells or culture medium by methods known in the art.
[00134] Heterologous proteins produced in ciliates can be targeted to either a constitutive secretory pathway or to a regulated secretory pathway.
[00135] Ciliates can engage in regulated secretion of proteins stored in cortical secretory organelles (granules) which are discharged in a stimulus-dependent or regulated fashion (17, Miller et al. (1990)). Dense core granules are specialized for stimulus-dependent secretory granules that function in exocytosis in ciliates. In Tetrahymena spp., these dense core granules are termed mucocysts, whereas the dense core granules of Paramecium tetraurelia are termed trichocysts (Hausmann (1978); Rosati and Modeo (2003)). [00136] Regulated secretion in ciliates can be triggered by the presence of chemical mediators known as secretagogues. For example, such mediators can cause increased levels of intracellular calcium (Ca2+), which, in turn, trigger fusion of cortical granules with the plasma membrane resulting in a release of the granule contents into the surrounding extracellular space. Examples of secretagogues useful in the invention include, but are not limited to, dibucaine, alcian blue and Ca2+ ionophores.
[00137] Mutant strains defective in the release of hydrolytic enzymes have also known in the art (Hunseler et al. (1992), Dev. Genet. 13: 167-173). In some embodiments, mutant strains lacking or exhibiting reduced levels of secreted hydrolytic proteases can be used for the production of surface expressed or secreted heterologous polypeptides.
[00138] Ciliates Useful in the Invention
[00139] The invention may be practiced with a variety of different ciliates. A ciliate as described herein can be any free-living ciliate, including but not limited to species in the genera Tetrahymena, Paramecium, Blepharisma, Colpidium, Euplotes, Stylonichia and Oxytricha. In some embodiments, the ciliate is a Tetrahymena spp.
[00140] The free-living ciliates are a large and diverse phylum (Ciliata) whose members display a structural and functional complexity comparable to that of higher metazoa (Frankel (2000), Meth. Cell Biol. 62: 27-125; Turkewitz et al. (2002), Trends in Genetics, 18, 35-40) and include over 7,000 species with 11 major subdivisions. Tetrahymenids and Paramecium belong to the Oligohymenophoreans.
[00141] Ciliates suitable for use with the methods described herein include, but are not limited to Tetrahymena thermophila, Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi, Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis, Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes
octocarinatus, Euplotes vannus, Monoeuplotes crassus, Blepharisma japonicus.
[00142] In some embodiments, the genetically modified ciliates described herein are genetically modified Tetrahymena spp., including T. thermophila.
[00143] Tetrahymena spp. are amenable to genetic manipulation, can be grown on a large scale and have a doubling time of 1.5-3 hrs. Unlike T. thermophila, which has an optimal growth temperature of 35°C, the optimal growth temperature for T. pyriformis is 34°C. Cells reach high-density in a short time on a variety of inexpensive media and can be expanded for growth in bioreactors up to several thousand liters in size (Hellenbroich et al. (1999); Appl. Microbiol. Biotechnol. 51 :447; de Coninck et al. (2000), J. Industr. Microbiol. Biotechnol. 24:285). Methods for transformation, along with robust, inducible promoters for driving high-level gene expression have recently been described for this system (Bruns and Cassidy-Hanley (2000), Methods Cell Biol. 62: 501-512; Gaertig andKapler (2000), Methods Cell Biol. 62: 486-500; Shang et al. (2002), Proc. Natl. Acad. Sci. U.S.A. 6, 3734-3739; Boldrin et al. (2006), Eukaryot Cell 2:422-425.
[00144] Some ciliates, including T. thermophila, exhibit nuclear dimorphism and contain two distinct types of nuclei in each cell. The micronucleus (MIC) is diploid and contains five pairs of transcriptionally inert chromosomes. The macronucleus is polyploid and functions as the transcriptionally active nucleus during vegetative growth. The micronucleus functions as the repository for genetic information for progeny produce by conjugation during sexual reproduction. During conjugation, the micronucleus undergoes meiosis to give rise to two pronuclei that are reciprocally exchanged between sexually mating cells. Upon fusion of the haploid gametes to produce a new zygotic micronucleus, the micronucleus undergoes two post-zygotic divisions resulting in the formation of new micronuclei and macronuclei. Upon formation of new macronuclei, the old macronuclei are resorbed and not transmitted to the sexual progeny. The cells then reproduce by asexual until the next round of conjugation.
[00145] Differentiation of a macronucleus (MAC) from the mitotic products of zygotic micronuclei produced upon sexual reproduction involves several programmed DNA rearrangements, including but not limited to (1) the deletion of internal eliminated sequences (IESs) from the MIC genome, (2) programmed site specific fragmentation of the five MIC chromosomes at specific chromosome breakage sequence (CBS) containing sites to form about 180 MAC chromosomes, and (3) amplification to increase the copy numbers of the newly formed MAC chromosomes.
[00146]
[00147] Polypeptides Having Increased Man3GlcNAc? Core N-glycans
[00148] The present invention provides compositions comprising a population of glycoproteins having predominantly or substantially uniform Man3GlcNAc2 core N-glycans. [00149] The advantages of producing glycoprotein compositions having a predominantly Man3GlcNAc2 core glycan include reduced presence of undesired glycoforms, less heterogeneous mixtures of glycoproteins, and/or higher concentrations of the more effective glycoform(s). Therefore, a pharmaceutical composition comprising glycoproteins having predominantly Man3GlcNAc2 core N-glycan can have beneficial features, including but not limited to, reduced immunogenicity, greater manufacturing consistency, and higher efficacy/potency.
[00150] In some embodiments, a glycoprotein of the present invention comprises at least one Man3GlcNAc2 core N-glycan structure at one or more Asparagine residues within the polypeptide. The glycoproteins are produced in a host cell that has been genetically engineered to produce a population of glycoproteins having Man3GlcNAc2 as the
predominant core N-glycan. In general, the genetically modified ciliates are transformed with one or more nucleic acids encoding the desired recombinant glycoprotein. In certain embodiments, the recombinant glycoprotein encoded by the nucleic acid is a therapeutic polypeptide (e.g., a cytokine, peptide hormone or antibody).
[00151] The genetically modified ciliates described herein are incubated under conditions suitable for producing the polypeptide. The ciliates can secrete the recombinant polypeptide into the culture medium either through a constitutive secretory pathway or a regulated secretory pathway.
[00152] The recombinant glycoprotein can then be separated from other components of the culture medium and be resuspended in a suitable vehicle to make the compositions.
[00153] The glycoproteins are produced in a host cell that has been genetically engineered to produce glycoproteins having predominantly or substantially uniform
Man3GlcNAc2 core N-glycans.
[00154] In embodiments where the recombinant glycoprotein is an
immunoglobulin molecule, the immunoglobulin molecule can comprise at least one
Man3GlcNAc2 glycan structure at Asn-297 of a CH2 domain of a heavy chain on the Fc region mediating immunoglobulin effector function in the immunoglobulin molecule. In another embodiment, the Man3GlcNAc2 glycan structure is on each Asn-297 of each CR2 region in a dimerized immunoglobulin. In another embodiment, the present invention provides compositions comprising immunoglobulins which are predominantly glycosylated with an N-glycan consisting essentially of Man3GlcNAc2 glycan structure at Asn-297.
Alternatively, one or more carbohydrate moieties found on an immunoglobulin molecule may be deleted and/or added to the molecule, thus adding or deleting the number of glycosylation sites on an immunoglobulin. Further, the position of the N-linked glycosylation site within the CH2 region of an immunoglobulin molecule can be varied by introducing asparagines (Asn) or N-glycosylation sites at varying locations within the molecule.
[00155] While Asn-297 is the N-glycosylation site typically found in murine and human IgG molecules (Kabat et al., Sequences of Proteins of Immunological Interest, 1991), the methods describe herein are not limited to Man3GlcNAc2 glycan structure at this site, nor does this site necessarily have to be maintained for function.
[00156] Glycosylation of the Fab region of an immunoglobulin can also occur in serum antibodies, for example at Asn-75 (Rademacher et al. (1986), Biochem. Soc. Symp. 51 : 131-148). Glycosylation in the Fab region of an immunoglobulin molecule is an additional site that can be combined in conjunction with N-glycosylation in the Fc region, or alone.
[00157] In one embodiment, a nucleic acid encoding the heavy and light chains of the immunoglobulin can be synthesized using overlapping oligonucleotides and each separately cloned into an expression vector for expression in a host cell.
[00158] In particular embodiments, the recombinant immunoglobulin encoded by the nucleic acid can be a humanized immunoglobulin.
[00159] While for many recombinant immunoglobulins the Man3GlcNAc2 will be linked to the nitrogen of the amide group of Asn-297, in particular embodiments, the site for the N-glycan linkage can be at an asparagine at a different site within the immunoglobulin molecule (other than Asn-297), or in combination with the N-glycosylation site in the Fab region.
[00160] The term antibody is used in the broadest sense and includes single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions which will bind to multiple epitopes or antigens. The terms specifically cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they contain or are modified to contain at least the portion of the CH2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the CR2 domain, or a variant thereof. Included within the term are molecules comprising the Fc region, Fc fusions and antibody- like molecules. Alternatively, these terms can refer to an antibody fragment of at least the Fab region that at least contains an N-linked glycosylation site. Included within the scope of the term "antibody" are classes of Igs, namely, IgG, IgA, IgE, IgM, and IgD. Also included within the scope of the terms are the subtypes of IgGs, namely, IgGl, IgG2, IgG3 and IgG4.
[00161] The term "Fc" fragment refers to the 'fragment crystallized' C-terminal region of the antibody containing the CR2 and CR3 domains. The term "Fab" fragment refers to the 'fragment antigen binding' region of the antibody containing the VH, CHI, VL and CL domains.
[00162] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a first species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they contain or are modified to contain at least one CR2.
[00163] "Humanized" forms of non-human {e.g., murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) which contain sequences derived from human immunoglobulins. An Fv fragment of an antibody is the smallest unit of the antibody that retains the binding characteristics and specificity of the whole molecule. The Fv fragment is a noncovalently associated heterodimer of the variable domains of the antibody heavy chain and light chain. The F(ab)'2 fragment is a fragment containing both arms of Fab fragments linked by the disulfide bridges. [00164] Polypeptides having a Man3GlcNAc2 N-glycan structure as described herein can also comprise fusion polypeptides.
[00165] Polypeptides produced by the genetically modified ciliates described herein can further include two or more polypeptides attached to one another by recombinant means. For example, in one embodiment, the polypeptide can be a fusion between a cytokine and an antibody. One skilled in the art will readily be capable of using recombinant DNA techniques to fuse an antibody reading frame with another therapeutic peptide (e.g., a cytokine). In certain embodiments, such fusion protein can further comprise a flexible linker sequence (for example a 5 -10 amino acid sequence composed primarily of alanine and glycine residues) such that the biological activity and folding of the fused polypeptides is maintained. In the case of full length antibody fusion proteins, one skilled in the art will recognize that that fusion of a polypeptide to the C-terminus of the full length antibody can be useful. One skilled in the art will also understand that both N and C terminal fusions are also possible in certain circumstances, for example where the fusion comprises a single chain antibody fragment (scFv).
[00166] Such fusion polypeptides can present combinations of biological activity and function that are not found in nature to create novel and useful molecules of therapeutic and industrial applications. Biological activities of interest include, but are not limited to, enzymatic activity, receptor and/or ligand activity, immunogenic motifs, and structural domains. Such fusion polypeptides are well known in the art, and the methods of creation will be well-known to those in the art.
[00167] Polypeptides having a Man3GlcNAc2 N-glycan structure as described herein can be variants of native polypeptides, wherein a fragment of the native polypeptide is used in place of the full length native polypeptide. In addition, pre-pro-, and pre-peptides can also be used in conjugation with the methods described herein. Variant polypeptides may be smaller in size that the native polypeptide, and may comprise one or more domains of a larger polypeptide. Also included are truncations of the polypeptide and internal deletions which may enhance the desired therapeutic effect of the polypeptide. Any such forms of a polypeptide is contemplated to be useful in the present invention provided that the desired biological activity of the polypeptide is preserved.
[00168] Pharmaceutical Compositions [00169] Polypeptides having Man3GlcNAc2 as the predominant N-glycan can be incorporated into pharmaceutical compositions wherein the polypeptide is an active therapeutic agent (See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The composition can depend on the intended mode of administration and therapeutic application. The composition can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[00170] Pharmaceutical compositions for parenteral administration can be sterile, substantially isotonic, pyrogen-free, sterile, and prepared in accordance with GMP of the U.S. Food and Drug Administration or similar body. The compositions can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. The compositions can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (See Langer (1990), Science 249: 1527 and Hanes (1997), Advanced Drug Delivery Reviews 28: 97-119.
[00171] Polypeptides or antibodies having a Man3GlcNAc2 N-glycan structure produced according to the methods described herein can be incorporated into pharmaceutical compositions comprising the polypeptide as an active therapeutic agent and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The form can depend on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent can be selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[00172] The polypeptides having a Man3GlcNAc2 N-glycan structure as the predominant N-glycan can also be incorporated into a variety of diagnostic kits and other diagnostic products such as an array.
[00173] The polypeptides having a Man3GlcNAc2 N-glycan structure as the predominant N-glycan can have many therapeutic applications for indications including, but not limited to, cancers, inflammatory diseases, infections, immune diseases, and autoimmune diseases.
[00174] The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
[00175] Example 1: Expressing a-l,2-mannosidase in a Ciliate
[00176] Described herein are genetically modified ciliates that express
glycoproteins having substantially uniform Man3GlcNAc2 core N-glycans or genetically modified ciliated that express glycoproteins having predominantly Man3GlcNAc2 core N- glycans. The Man3GlcNAc2 glycan is native to T. thermophila and other ciliates and therefore is not deleterious to engineered cells. Man3GlcNAc2 provides a native glycan platform for complex glycan synthesis in ciliates that is quite distinct from the non-native MansGlcNAc2 glycan generated in previous efforts in yeast. Unlike yeast (which, contain a cell wall composed of a mixture of carbohydrates and proteoglycans including β 1 ,3 and β 1 ,4 glucans, mannoproteins and chitin, and which is essential to the structural integrity of the cell), expression of heterologous a-l,2-mannosidase does not adversely affect the health of the cell. The removal of Ochl activity leads to a substantial loss of cell wall mannose content in the form of mannoproteins that results in a structurally compromised cell. This is evidenced by characteristic cell- wall deficiency phenotypes in Ochl mutants of a number of different yeast species such as cell-aggregation, bloating and slow growth. Thus protein expression can be severely compromised in a fungal organism with this genetic background.
[00177] In one aspect, the methods described herein relate to ciliate cell lines that produce uniform Man3GlcNAc2 glycans that are wild-type except for expression of a heterologous a-l,2-mannosidase. An expression cassette was designed to express an a- 1,2- mannosidase (GenBank Gene ID, 3408991) from the ciliate Entamoeba histolytica, in the ciliate Tetrahymena thermophila (Figure 6). The expression cassette contains a
cycloheximide resistance cassette that incorporates the rpl29 gene and a promoter derived from the Tetrahymena thermophila catalase gene (GenBank Gene ID, 7837249) upstream of the E. histolytica a-l,2-mannosidase. The expression cassette is flanked by approximately 1Kb of DNA homologous to the Tetrahymena BTU1 gene locus to direct homologous recombination at that locus. In this particular instance the 3' BTU targeting DNA also acts as a terminator for expression of the a-l,2-mannosidase gene. The choice of promoter, terminator, resistance cassette and chromosomal targeting locus are only meant to illustrate the method of expression of an a-l,2-mannosidase in T thermophila. It is well understood that one skilled in the art could achieve a similar outcome by employing interchangeable transcriptional, selection and chromosomal targeting elements. Transformation of the expression cassette into wild-type T thermophila cells results in cycloheximide -resistant strains that express the heterologous a-l,2-mannosidase as judged by western analysis and immunofluorescence (Figure 7). Integration of the expression cassette into either the macronucleus or micronucleus of ciliates is achievable through established methods in ciliate-based molecular biology. In this particular instance immunofluorescence suggests that the native E. histolytica mannosidase sequence is sufficient to target the protein to the Tetrahymena ER (Figure 7).
[00178] Example 2: Alg 11 Deficient Ciliates [00179] Uniform Man3GlcNAc2 glycans can also be produced from ciliates by generating a strain that is deficient in the Algl 1 gene product. Algl 1 is an a- 1,2- mannosyltransferase that sequentially adds a 1 ,2 linked mannose residues to the lipid-linked glycan precursor on the cytoplasmic face of the ER. The lipid-linked MansGlcNAc2 glycan precursor is then translocated to the luminal face of the ER where three glucose residues are added sequentially to the terminal a 1 ,2 linked mannose residue prior to transfer to protein.
[00180] Algl 1 is an essential gene in a majority of eukaryotic cells. Since ciliates, such as Tetrahymena have a ploidy of 45 gene copies in their macronucleus, generating a strain that is deficient in the Algl 1 gene product can be used as a strategy to produce uniform Man3GlcNAc2 glycans. In ciliates, phenotypic assortment following introduction of a disruption cassette can result in a gene dosage effect where a majority of glycans are produced as Man3GlcNAc2 structures.
[00181] The Tetrahymena macronucleus has a ploidy of 45 therefore in the context of a knockout, to ensure that the native locus has been completely replaced by the knockout cassette, cells can be "pushed" in increasing levels of drug so that the higher the tolerence to drug the more copies of the knockout cassette containing the drug resistance marker have replaced the native allele. Thus, cells can be pushed by increasing levels of selective pressure {e.g., the concentration of a selective drug). Cells phenotypically assorted in increasing concentrations of selection drug exhibited a slow growth rate phenotype, indicating of the importance of this gene to cell health. The effect on the glycan profile of pushed Algl 1 knockout strains shows that the pool of Man3GlcNAc2 is increased substantially compared to wild type cells (71%:30-45% total glycans, respectively) (Figure 9). Complete phenotypic assortment results in the complete replacement of all 45 copies of the native allele - this is possible when the gene being knocked out is non-essential. The fact that full phenotypic assortment was not obtained by drug pushing shows that Algl 1 is an essential gene. Instead, the cells were observed to tolerate a threshold drug concentration that the cells could tolerate indicating that these "pushed" cells retained the minimum number of native Algl 1 alleles required for viability. This kind of genetic depletion manifests in a biochemical phenotype and shows that this approach is useful in ciliates for the production of uniform Man3GlcNAc2 glycans.
[00182] Any approach known in the art that is suitable for reducing, or eliminating Algl 1 function in a ciliate can be used in conjunction with the methods described herein. An exemplary approach includes, but is not limited to, introducing random mutations to cells containing an Algl 1 knockout allele(s). Strains of such cells can then be screened to identify strains that have increased tolerance for the knockout, are healthier or produce a larger relative pool of Man3GlcNAc2 glycans.
[00183] Example 3: Alg 11 Deficient Ciliates
[00184] It is possible to produce varying quantities of Man3GlcNAc2 glycans in genetically modified fungal microorganisms that normally produce Glc3Man9GlcNAc2 N- glycan precursors. Late stages of N-glycan precursor synthesis can be interrupted through deletion of the ALG3 gene to keep N-glycan precursor intermediates from becoming larger than Man5GlcNAc2. In one embodiment, this approach can be performed in a strain background that additionally does not produce N-glycan outer chain mannan in the Golgi. Such strains that do not produce N-glycan outer chain mannan in the Golgi can be generated by deletion of ochl. The Man5GlcNAc2 glycan can become transferred to proteins and converted to a Man3GlcNAc2 glycan through the action of an in vivo expressed a 1,2- mannosidase.
[00185] In the yeasts S. cerevisiae, P. pastoris and H. polymorpha, cells deficient in both alg3 and ochl do not yield homogeneous Man5GlcNAc2 glycans (Davidson et al. (2004), Glycobiology 14:399; Verostek et al. (1991), J. Biol. Chem. 266:5547; Oh et al. (2008), Biotechnol. J. 3:659). Instead, the observed glycans include Man5GlcNAc2 along with larger glycoforms {e.g., Hex6-12GlcNAc2), some of which are recalcitrant to a 1,2- mannosidase digestion. P. pastoris and S. cerevisiae cells also display impaired growth compared to wild type cells (Davidson et al. (2004), Glycobiology 14:399; Verostek et al. (1991), J Biol Chem. Mar 25;266(9):5547-51) whereas H. polymorpha cells do not (Oh et al. (2008), Biotechnol. J. 3:659).
[00186] In H. polymorpha, in vivo expression of an a 1 ,2-mannosidase in the alg3 ochl background yielded predominantly Man3GlcNAc2 glycans.
[00187] Deletion of the AlgC gene (synonymous to yeast Alg3) in filamentous Apsergillus fungi yields N-glycans from Man3_5GlcNAc2 (Kainz et al. (2008) Appl. Environ. Microbiol. 74: 1076).
[00188] This methodology can be applied to generating Man3GlcNAc2 in fungi, however results can vary from organism to organism. In contrast to fungal glycoengineering, ciliate cells naturally lack an ALG3 gene and produce and process Man5GlcNAc2 to
Man3GlcNAc2 without any engineering.
[00189] However, the Algl 1 gene in Tetrahymena encodes a a 1,2- mannosyltransferase. Algl 1 deficient strains will be generated to eliminate a- 1,2- mannosyltransferase thereby generating a genetically modified ciliate predominantly expressing polypeptides or antibodies having a Man3GlcNAc2 N-glycan structure. In one embodiment, a a 1 ,2-mannosidase can further be expressed on the ER of such proteins to enhance the ability of a ciliate to form Man3GlcNAc2 precursors.
[00190] Example 3: Evaluation of N-Glycan Content of Secreted
Glycoproteins
[00191] Evaluation of N-glycan content of secreted glycoprotein's was determined for wild-type and engineered strains expressing a-l,2-mannosidase in the following way:
[00192] T. thermophila strains were cultured in 100 ml Neff (5% glucose, 2.5 g/L Bactopeptone, 2.5 g/L yeast extract). Spent culture medium (SCM) was collected by centrifugation at 3500 x g for 10 minutes and then concentrated 100 fold in Vivaspin 20 columns (Sartorius) containing a 30 kDa molecular weight cut-off (MWCO) membrane. Concentrated SCM was washed with 10 mM Tris-Cl pH 8.0 in the same columns and sample proteins were purified by anion-exchange chromatography. Concentrated and buffer exchanged SCM was loaded onto a 600 μΐ Q-sepharose column pre-equilibrated with 10 mM Tris-Cl, pH 8.0 buffer. The resin was washed with 2-ml 10 mM Tris-Cl, pH 8.0 and then with 2-ml 10 mM Tris-Cl, 0.15M NaCl, pH 8.0. Bound proteins were eluted with 3 ml 10 mM Tris-Cl, 1 M NaCl, pH 8.0, and concentrated 10-fold in a Vivaspin 20 column (Sartorius) containing a membrane with a 10 kDa MWCO. Proteins were denatured by boiling in the presence of SDS and then treated with 20,000 units of N-Glycosidase F (PNGase F, New England Biolabs) to release N-linked glycans. Samples containing released N-glycans were desalted on 200 μΐ of a mixed-bed resin (AG 501-X8) and purified on porous graphitized columns (Hypersep Hypercarb, Thermo Scientific). Samples were dried in a speed-vacuum at room temperature and analyzed by high-pressure anion exchange chromatography (HPAEC).
[00193] Quantitative HPAEC analysis showed that expression of an a- 1,2- mannosidase in engineered cells increased the N-linked Man3GlcNAc2 pool to over 80% total N-glycan compared to approximately 30-45%) in wild-type cells (Figure 8). [00194] In certain embodiments, the methods described herein can be further modified to increase the linked Man3GlcNAc2 pool to over 80% of total N-glycan content.
[00195] For example, in one embodiment the a-l,2-mannosidase expression cassette copy number can be increased. In another embodiment, a more efficient native promoter or a more efficient heterologous promoters can be used in conjunction with the methods described herein. In still a further embodiment, the methods described herein can be performed using a more active a-l,2-mannosidase gene.
[00196] Additional References
[00197] 1. Pavlou and Reichert (2004), "Recombinant protein therapeutics-success rates, market trends and values to 2010," Nat. Biotechnol. 22: 1513-1519.
[00198] 2. Langer, (2005), 3rd Annual Report and Survey of Biopharmaceutical Manufacturing, Capacity and Production. BioPlan Associates, Inc.
[00199] 3. Sethuraman and Stadheim (2006), "Challenges in therapeutic glycoprotein production," Curr. Opin. Biotechnol. 17:341-346.
[00200] 4. Jefferis (2005), "Glycosylation of Recombinant Antibody
Therapeutics," Biotechnol. Prog. 21 : 11-16.
[00201] 5. Logtenberg (2007), "Antibody cocktails: next generation
biopharmaceuticals with improved potency," Trends Biotechnol. 25:390-394.
[00202] 6. Kornfeld and Kornfeld (1985), "Assembly of asparagine-linked oligosaccharides," Annu. Rev. Biochem. 54:631-664.
[00203] 7. Fan et al. (1997), "Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding," Eur. J. Biochem. 246:243-251.
[00204] 8. Sitia and Braakman (2003), "Progress quality control in the endoplasmic reticulum protein factory," Nature 426:891-894
[00205] 9. Skropeta (2009), "The effect of individual N-glycans on enzyme activity," Bioorg. Med. Chem. 17:2645-2653. [00206] 10. Rudd et al. (2001), "Glycosylation and the immune system," Science 291 :2370-2376.
[00207] 11. Helenius and Aebi (2001), "Intracellular functions of N-linked glycans," Science 291 :2364-2369.
[00208] 12. Sinclair and Elliot (2005), "Glycoengineering: The effect of glycsylation on the properties of therapeutic proteins," J. Pharm. Sci. 94: 1626-1635.
[00209] 13. Hoffmeister et al. (2003), "Glycosylation restores survival of chilled blood platelets," Science 301 : 1531-1534.
[00210] 14. Shantha Raju (2008), "Terminal sugars of Fc glycans influence antibody effector functions of IgGs," Curr. Opin. Immun. 20:471-478.
[00211] 15. Nimmerjahn and Ravetch (2008), "Fc gamma receptors as regulators of immune responses," Nat. Rev. Immunol. 8:34-47.
[00212] 16. Jeffries and Lund (2002), "Interaction sites on human IgG-FC for FcgammaR: current models," Immunol. Lett. 82:57-65.
[00213] 17. Mimura et al. (2001), "Role of oligosaccharide residues of IgGl-Fc in Fc Rllb binding," J. Biol. Chem. 276:45539-45547.
[00214] 18. Jigami (2008), "Yeast glycobiology and its application," Biosci. Biotechnol. Biochem. 72:637-648.
[00215] 19. Gerngross (2004), "Advances in the production of human therapeutic proteins in yeasts and filamentous fungi," Nat. Biotechnol. 22: 1409-1414
[00216] 20. Li et al. (2006), "Optimization of humanized IgGs in glycoengineered Pichia pastoris," Nat. Biotechnol. 24:210-215.
[00217] 21. Hamilton et al. (2006), "Humanization of yeast to produce complex terminally sialylated glycoprotein' s," Science 313: 1441-1443.
[00218] 22. Eisen et al. (2006), "Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote," PLoS Biol. 4: 1620-1642.
[00219] 23. Coyne et al. (2008), "Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure," BMC Genomics 9:562. [00220] 24. Taniguchi et al. (1985), "Carbohydrates of Lysosomal enzymes secreted by Tetrahymena pyriformis." J. Biol. Chem. 260:13941-13946.
[00221] 25. Weide et al. (2006), "Secretion of functional human enzymes by Tetrahymena thermophila, " BMC Biotechnol. 6: 19.
[00222] 26. Becker and Rusing (2003), "Structure of N-glycosidic carbohydrates of secretory proteins of Tetrahymena thermophila, " J. Eukaryot. Microbiol. 50:235-239.
[00223] 27. Banno et al. (1987), "Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis, " Exp. Cell Res. 170:259-268.
[00224] 28. Blum (1976), "Lysosomal hydrolase secretion by Tetrahymena: a comparison of several intralysosomal enzymes with the isoenzymes released into the medium," J. Cell Physiol. 89:457-72.
[00225] 29. Debono and Gordee (1994), "Antibiotics that inhibit fungal cell wall development," Annu. Rev. Microbiol. 48:471-497.

Claims

WHAT IS CLAIMED IS:
A genetically modified ciliate that expresses glycoproteins having predominantly Man3GlcNAc2 core N-glycans.
A genetically modified ciliate that expresses glycoproteins having predominantly Man3GlcNAc2 core N-glycans, wherein the ciliate has significantly increased a- 1,2- mannosidase activity.
A genetically modified ciliate that expresses glycoproteins having predominantly Man3GlcNAc2 core N-glycans, wherein the ciliate expresses a heterologous sequence encoding a protein having a-l,2-mannosidase activity.
The genetically modified ciliate of claim 3, wherein the protein having a- 1,2- mannosidase activity comprises an endoplasmic reticulum targeting sequence.
The genetically modified ciliate of claim 3, wherein the protein having a- 1,2- mannosidase activity comprises a signal sequence.
The genetically modified ciliate of claim 3, wherein the protein having a- 1,
2- mannosidase activity comprises a sequence selected from the group consisting of: MKFNILIILIISLFINELRA (SEQ ID NO: 1), MNKFSLVLCLSLVLISLRAES (SEQ ID NO: 2), MSKLLIAVLLCLAIITPTVLC
(SEQ ID NO: 3),
MRS STLSILLLCILGASI A
(SEQ ID NO: 4), MQINRVIFVVSALIAVSLLTGFA
(SEQ ID NO: 5), MQNKTIIICLIISQLLVSVFSSAGGQA
(SEQ ID NO: 6),
MNTKLLIALPILALLSIGAVFL (SEQ ID NO: 7),
MNKTLILALVGVLALTATTLVA (SEQ ID NO: 8), MNKLVLIALVTLFAGVMA (SEQ ID NO: 9), MQKSIIIAAILLVGLASA (SEQ ID NO: 10),
MNKTSIILIASILSAALCGA (SEQ ID NO: 11), MNKVALIASFLAGLSILSIS (SEQ ID NO: 12), MKTQFVFLFTLLLLNALC (SEQ ID NO: 13),
MSKLQFVLIAALLLVAVSA (SEQ ID NO: 14), MNQKQLFALLVIVFIQISSTSC (SEQ ID NO: 15), MKKI ALL S VC AFILLI SF ANC (SEQ ID NO: 16), and
MAALKILLVAILLCSGCLS (SEQ ID NO: 17)
7. The genetically modified ciliate of any of claims 3-6, wherein the protein having a-
1 ,2-mannosidase activity is selected from the group consisting of Aspergillus saitoi - 1 ,2-mannosidase, Trichoderma reesei a-l,2-mannosidase, Penicillium citrinum a- 1,2- mannosidase, Aspergillus nidulans a-l,2-mannosidase, Homo sapiens a- 1,2- mannosidase IA, Homo sapiens IB 1 ,2-a-mannosidase, Lepidopteran insect Type I a- 1 ,2-mannosidase, Homo sapiens a D mannosidase, Xanthomonas a-l,2-mannosidase Mouse IB a-l,2-mannosidase, and Bacillus sp. a-l,2-mannosidase.
8. A genetically modified ciliate that expresses glycoproteins having predominantly Man3GlcNAc2 core N-glycans, wherein the ciliate has significantly decreased a- 1,2- mannosyltransferase activity.
9. A genetically modified ciliate that expresses glycoproteins having predominantly Man3GlcNAc2 core N-glycans, wherein the ciliate has a micronuclear genotype comprising one or more non- functional Algl 1 alleles.
10. The genetically modified ciliate of claim 8 or 9, comprising a modification selected from the group consisting of a deletion, an insertion, a substitution or an inversion in an Algl 1 allele.
11. The genetically modified ciliate of any one of claims 1-10, wherein the ciliate is
selected from the group consisting of Tetrahymena thermophila,
Tetrahymena pyriformis, Tetrahymena paravorax, Tetrahymena hegewischi,
Tetrahymena capricornis, Tetrahymena canadensis, Tetrahymena borealis,
Paramecium tetraurelia, Paramecium caudatum, Polytomella agilis, Stylonichia lemnae, Oxytricha granulifera, Euplotes aediculatus, Euplotes focardii, Euplotes octocarinatus, Euplotes vannus, Monoeuplotes crassus, Blepharisma japonicus..
12. A method for producing a recombinant glycoprotein having predominantly
Man3GlcNAc2 core N-glycans, the method comprising: (a) transforming a ciliate of any one of claims 1-11 with an expression vector encoding the recombinant glycoprotein,
(b) culturing the ciliate under conditions which promote expression of the recombinant glycoprotein, and
(c) isolating the recombinant glycoprotein.
13. The method of claim 12, wherein the recombinant glycoprotein is a human protein.
14. The method of claim 12, wherein the recombinant glycoprotein is a therapeutic protein.
15. The method of claim 12, wherein the recombinant glycoprotein is a cytokine.
16. The method of claim 12, wherein the recombinant glycoprotein is a fusion protein.
17. The method of claim 12, wherein the recombinant glycoprotein is an antibody.
PCT/US2011/029229 2010-03-22 2011-03-21 Production of glycoproteins in genetically modified ciliates WO2011119498A1 (en)

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