WO2013066765A1 - Mutation de tup1 dans de la levure glycomodifiée - Google Patents

Mutation de tup1 dans de la levure glycomodifiée Download PDF

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Publication number
WO2013066765A1
WO2013066765A1 PCT/US2012/062231 US2012062231W WO2013066765A1 WO 2013066765 A1 WO2013066765 A1 WO 2013066765A1 US 2012062231 W US2012062231 W US 2012062231W WO 2013066765 A1 WO2013066765 A1 WO 2013066765A1
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tupl
polypeptide
host cell
polynucleotide
isolated
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PCT/US2012/062231
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Robert Davidson
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Merck Sharp & Dohme Corp.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to an isolated polynucleotide comprising a Tupl 070 allele as well as fungal host cells comprising such an allele.
  • Methods for producing heterologous polypeptides, such as immunoglobulins, using host cells comprising such alleles are also part of the present invention.
  • Therapeutic monoclonal antibodies are key products for the biopharmaceutical industry. Frequently, therapeutic mAbs are produced in mammalian cells, for example in Chinese hamster ovary (CHO) cell lines. The glycan profiles of recombinant antibodies produced in mammalian cells can be highly heterogeneous.
  • Heterogeneity can vary widely from clone to clone and is dependent on the mode of production and culture conditions .
  • An antibody' s glycan profile can have a significant effect on ADCC (antibody- dependent cellular cytotoxicity) and CDC (complement- dependent cytotoxicity) .
  • Alternate expression systems whose genetics facilitate control over glycosylation would be beneficial.
  • Yeast such a Pichia constitute one such system.
  • the glycosylation profile in particular the level of glycan
  • Pichia cell uniformity, exhibited by a given Pichia cell can be modulated by over-expression or under-expression (e.g., knock out) of certain genes .
  • the present invention provides an isolated polynucleotide that encodes a Pichia pastoris tupl TM allele (e.g., SEQ ID NO: 3 or nucleotides 1-411 or 1-414 thereof) ; a Saccharomyces cerevisiae t plTM allele; a Candida albicans tupl CTD allele or a Kluyveromyces lactis tupl allele; or a vector comprising said polynucleotide,- e.g., amino acids about 1 to about 137 of SEQ ID NO: 2; amino acids about 1 to about 139 of SEQ ID NO: 5; amino acids about 1 to about 120 of SEQ ID NO: 7; or amino acids about 1 to about 112 e.g.
  • a Pichia pastoris tupl TM allele e.g., SEQ ID NO: 3 or nucleotides 1-411 or 1-414 thereof
  • the present invention also includes an isolated polypeptide encoded any such polynucleotide.
  • the present invention also includes an isolated fungal host cell comprising any such polynucleotide or vector comprising the polynucleotide or polypeptide encoding such a polynucleotide; e.g. , wherein the polynucleotide is chromosomally integrated.
  • the host cell comprises a heterologous
  • polynucleotide and/or polypeptide e.g., an immunoglobulin; for example of an antibody or antigen-binding fragment thereof that bind specifically to an antigen selected from the group consisting of: VEGF, HER1 , HER2 , HER3 , glycoprotein Ilb/lIIa, CD52 , IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3 Receptor, alpha-4 (alpha 4) integrin, PCSK9,
  • an immunoglobulin for example of an antibody or antigen-binding fragment thereof that bind specifically to an antigen selected from the group consisting of: VEGF, HER1 , HER2 , HER3 , glycoprotein Ilb/lIIa, CD52 , IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR)
  • immunoglobulin E igE
  • RSV F protein ErbB2, VEGF, HER1, HER2 , HER3, glycoprotein Ilb/IIla, CD52 , IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3 Receptor, alpha-4 ⁇ alpha 4) integrin, PCS 9, immunoglobulin E (IgE), RSV F protein and ErbB2; or wherein the heterologous polypeptide is an immunoglobulin chain of an antibody or antigen-binding fragment thereof that is selected from the group consisting of: Abciximab; Adalimumab; Alemtuzumab; Basiliximab; Bevacizumab; Cetuximab;
  • Certolizumab Daclizumab; Dalotuzumab; Denosumab; Eculizumab;
  • Efalizumab Efalizumab; Gemtuzumab; Ibritumomab tiuxetan; Infliximab;
  • Muromonab-CD3 Natalizumab; Omalizumab; Palivizumab; Panitumumab; Ranibizumab; Rituximab; Tositumomab; and Trastuzumab.
  • the present invention further comprises a method for
  • an isolated Pichia pastoris host cell comprising mutating endogenous chromosomal Tupl in an isolated fungal host cell, e.g., a Pichia pastoris cell, wherein said mutated Tupl encodes a polypeptide comprising amino acids 1-137 of Pichia pastoris Tupl and/or introducing a polynucleotide that encodes said polypeptide or a homologue thereof ⁇ e.g., S. cerevisiae tuplTM, C. albicans tupl CTD or K.lactis tupl CTD ) into an isolated Pichia pastoris cell.
  • the present invention also provides a method for producing a heterologous polypeptide comprising introducing a heterologous polynucleotide encoding said polypeptide (e.g., an immunoglobulin as discussed herein) into an isolated fungal host cell as discussed herein which comprises a tupl TM allele and culturing said cell under conditions where the heterologous polypeptide is expressed in said cell.
  • a heterologous polypeptide e.g., an immunoglobulin as discussed herein
  • the heterologous polypeptide is secreted from the host cell and/or purified from said cell and/or cellular growth medium.
  • FIG. 1 Alignment of Tupl proteins from P. pastoris, 3. cerevisiae, and C. albicans.
  • the protein sequences of P. pastoris Tuplp (CCA39141.1) , S. cerevisiae Tuplp (AAA34413 , 1) , and C. albicans Tuplp (P0CY34.1) were obtained from Genbank and aligned with the ClustalV algorithm using Lasergene Megalign software (DNAstar, Madison, WI) .
  • Figure 2 Graphical lineage of glycoengineered Pichia strains .
  • Strain N RL-Y11430 is a wild P.
  • GFlx.y indicates the glycoform produced by each strain as depicted in Figure 7. Auxotrophic loci for each strain, if any, are listed. The strains containing the spontaneous tupl -2 SNV are indicated by the transparent gray oval .
  • Figure 3 Identification and location of the spontaneous tupl -2 single nucleotide variant.
  • YGLY8292, YGLY8323, and YGLY12501 are depicted with the location of the S V indicated by an arrow.
  • B A UCSC genome browser
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coll.
  • the plasmid contains the pUCl9 Ori and AmpR/KanR region for E . coli maintenance as well as the P.
  • Figure 5 Restriction, map of plasmid p6LY5640.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • Figure 6 Restriction map of plasmid pGLY5883.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • Figure 7 Depiction of the glycoforms generated by P. pastoris glycoengineered strains . Stepwise glycoengineering of P. pastoris yeast strains results in the modification of the N-glycosylation machinery. The common N-glycans generated by members of the strain lineage shown in
  • Figure 2 are depicted.
  • the pink ribbon represents the protein backbone.
  • the individual sugar residues are represented as follows: circle, GlcNAc; diamond, galactose; star, NANA/sialic acid, open square, a-1, 4-mannose,- solid square, a-l, 6-mannose;
  • FIG. 8 The P. pastoris TUPl rollin/rollout complementation strategy.
  • the plasmid pGLY5640 containing the TUPl wild type allele was digested with Jipal and transformed into strain YGLY16676, which is tupl-1 and expresses the aHER2 antibody, resulting in strain
  • YGLY19192 which has a duplicated TUPl locus containing the mutant and wild type alleles.
  • Counterselection on 5-Fluoro Orotic Acid which selects for removal of URAS along with one of the two TUPl alleles, followed by screening via DNA sequencing, yielded strain YGLY19193, containing only the TUPl wild type allele.
  • the URAS gene is then reinserted to generate strains YGLY19250 and
  • Figure 9 Restriction map of plasmid pGLY579.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the pUC19 backbone, the P. pastoris HISS 5' and ORF as well as the HIS3 3' region for integration, and the lacZ-URAS-lacZ blaster as a selectable marker (Nett, 2005) ,
  • the vector is digested with Sfil to release the pUC19 sequence and linearize the vector, thus promoting integration at the HIS3 locus, and selected for in the absence of uracil.
  • tupl* (tupl-1) aHER2 expressing strain YGLY13992 and TUPl WT aHER2 expressing strains YGLY19250 and YGLY19251 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction.
  • mAb was quantified by HPLC and reported as mg/L. Purified protein was also analyzed by Caliper GXII under denatured non-reducing and denatured reducing ⁇ with 1M DTT) conditions.
  • Figure 11 MALDI-TOF MS of N-glycans from purified mAb of bupl-1 mutant and TUP1 wild type complemented strains.
  • tupl* (tupl-1) aHER2 expressing strain YGLY13992 and TUP1 T HER2 expressing strain YGLY19250 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction. Following protein A purification, protein was deglycosylated by PNGase digestion and filter purified N-glycans were subjected to MALDI-TOF MS.
  • Figure 12 Restriction map of plasmid pGLY167b.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the pUC19 backbone, the P. pastoris ARG1 5' and ARG1 3' regions for
  • the plasmid also contains expression cassettes with P. pastoris GAPDH promoter-driven Drosophila melanogaster Mannosidase II and P. pastoris PMAl promoter-driven Human GlcNAc Transferase II, which are required for the maturation the -l,6 arm of complex N-linked glycans.
  • P. pastoris For introduction into P. pastoris the vector is digested with Sfil to release the pUCl9 sequence and linearize the vector, thus promoting integration at the ARG1 locus, and selected for on media lacking histidine.
  • Figure 13 Restriction map of plasmid pGLY8135.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • TUPl wild type (YGLY4829) and tupl-1 mutant (YGLY20599) strains from parallel lineages of GFI5.0 glycoengineered strains were each transformed with the pGLY8135 ARG1 marked ccHER2
  • Figure 15 Restriction map of plasmid pGLY8129.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the mutant tupl-1 (Ql38stop) P. pastoris TUPl gene amplified by PGR and sequence verified in the pCR2.1 TOPO cloning vector (Invitrogen, Carlsbad, CA) .
  • Figure 16 Restriction map of plasmid pGLY81 9.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • pastoris URA5 selectable marker and mutant tupl-1 (Ql38stop) P. pastoris TUPl gene For introduction into P. pastoris the vector is digested with Hpal or Bglll to linearize the vector, thus promoting integration at the TUPl locus, and selected for in the absence of uracil.
  • Figure 17 The P. pastoris tupl-1 rollin/rollout mutation re- introduction strategy.
  • the plasmid pGLY8149 containing the tupl-1 mutant allele was digested with Hpal and transformed into strain YGLY19193, which is TUPl wild type and expresses the aHER2 antibody, resulting in strain YGLY23502, which has a duplicated TUPl locus containing both the mutant and wild type alleles.
  • Counterselection on 5-Fluoro Orotic Acid which selects for removal of URA5 along with one of the two TUPl alleles, followed by screening via DNA sequencing, yielded strain YGLY21058, containing only the tupl-1 mutant allele.
  • the URA5 gene is then reinserted to generate strains YGLY26468 and YGLY26469.
  • CS counterselection.
  • YGLY26468 TUPl WT (XHER2 expressing strains YGLY19250 and YGLY19251 as well as TUPl/tupl* heterozygous strains YGLY19192 and YGLY23502 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction.
  • A Following protein A purification, purified protein was analyzed by Caliper GXII under denatured non-reducing
  • Figure 19 MALDI-TOF MS of N-glycans from purified mAb of GFI5.0 TtJPl wild type complemented and fcupl-2 mutant reconstituted.
  • the GFI5.0 TUPl wild type complemented aHER2 expressing strain YGLY19250, TUPl/tupl* heterozygous strain YGLY23502 and tupl* mutant reconstituted strain YGLY26468 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction. Following protein A purification, protein was deglycosylated by PNGase digestion and filter purified N-glycans were subjected to MALDI-TOF MS.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • Figure 21 Restriction map of plasmid pGLY9894.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • P. pastoris RPL10 promoter ⁇ driven by the P. pastoris RPL10 promoter
  • P. pastoris AOXl promoter cassette containing the P. pastoris TUPl full length wild type gene flanked by EcoRl/Fsel restriction sites.
  • the vector is digested with Spel to linearize the vector, thus promoting integration at the URA2 locus, and selected for on medium containing l-3mM sodium arsenite .
  • Figure 22 Restriction map of plasmid pGLY9895.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • the yeast pastoris URA6 gene for integration the S. cerevisiae ARR3 gene (driven by the P. pastoris RPL10 promoter) as a selectable marker, and a P. pastoris AOXl promoter cassette containing the P. pastoris tupl-l mutant (1-137) gene flanked by EcdRl/FseX restriction sites.
  • the vector is digested with Spel to linearize the vector, thus promoting integration at the URA2 locus, and selected for on medium containing l-3mM sodium arsenite.
  • Figure 23 Comparison of antibody productivity in GFI5.0 TUPl wild type complemented strains overexpressing the TUPl wild type or tupl-l mutant allele.
  • tupl* (tupl-l) CXHER2 expressing strain YGLY13992 and TUPl wild type (XHER2 expressing strain YGLY19250 as well as YGLY19250- derived strains expressing AOXl-driven cassettes of either wild type TUPl or mutant tupl* 1-137 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction. Following protein A purification, purified protein was analyzed by Caliper GXII under denatured non-reducing conditions. For each sample, the area under the curve was quantified based on a mAb protein standard and is reported as mg/L of the original Micro24 fermentation broth volume.
  • Figure 24 Comparison of antibody productivity in GFI2.0 TUP1 wild type strains overexpressing the TUPl wild type or tupl-l mutant allele.
  • TUPl wild type ⁇ xHER2 expressing strain YGLY4140 as well as YGLY 140 -derived strains expressing AOXl-driven cassettes of either wild type TUPl or mutant tupl* 1-137 were cultivated in Applikon 5ml minibioreactors in a 72h methanol induction. Following protein A purification, purified protein was quantified by reverse phase HPLC, using the area under the curve based on a mAb protein
  • Figure 25 Comparison of antibody productivity in GFI2.0 TUPl wild type strains overexpressing the TUPl wild type or tupl-l mutant allele at 1L fermentation scale.
  • TUPl wild type aHER2 expressing strain YGLY4140 as well as YGLY4140 -derived strains expressing AOXl-driven cassettes of either wild type TUPl or mutant tupl* 1-137 were cultivated in 1L Dasgip fermenters for -80 +/-5h of methanol induction. Following protein A purification, purified protein was quantified by reverse phase HPLC, using the area under the curve based on a mAb protein
  • Figure 26 Restriction map of plasmid pGLY4362.
  • the E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli.
  • the plasmid contains the P.
  • TUP1 wild type glycosylated human Insulin expressing strain YGLY25818 as well as YGLY25818 -derived strains expressing AOX1 -driven cassettes of either wild type TUP1 (YGLY26470) or mutant tupl* 1-137 (YGLY26472, YGLY26473) were cultivated in 1L Dasgip fermenters for -80 +/-5h of methanol induction. Following cultivation, supernatant was separated on reducing SDS-PAGE.
  • Insulin protein was identified by Q-ToF mass spectrometry analysis and is marked by an arrow.
  • Figure 28 Western blot of TUP1, tupl* and tuplA strains probed with an anti -PpTUPl peptide antibody
  • TUP1 wild type ⁇ both parental yll430 and glycoengineered GFI2.0
  • tuplA mutant, tupl* mutant, tupl* + TUP1 complemented both roll -in and KINKO knock- in ⁇ and A0X1-TUP1 or AOXl-tupl* overexpressing strains were cultivated in shake flasks in glycerol and methanol and whole extracted protein was separated on SDS-PAGE and subjected to Western analysis with a guinea pig anti-TUPl peptide antibody followed by an HRP labelled mouse anti-guinea pig secondary Ab. Full length Tuplp is marked by an arrow. The mutant tupl-l protein is not detected.
  • G glycerol; , methanol.
  • YGLY5828 (see Figure 2 for strain lineage) were struck for singles on YSD medium and YSD medium containing 0.2M CaCl 2 . Plates were incubated for 5 days at 24 °C then photographed.
  • Figure 30 Complementation of the tupl -2 allele in a
  • glycoengineered strain results in Ca 2+ sensitivity
  • Next-generation Genome Sequencing ⁇ NGS of glycoengineered Pichia strains has revealed a useful mutation, present in a GFI5.0 cell line, which promotes recombinant expression of monoclonal antibodies (mAb) and other polypeptides with high productivity and reproducibly high N-glycan uniformity.
  • This mutation is a single nucleotide variant nonsense mutation that results in a C-terminal truncation of a gene (Pp03g 016900) encoding a homolog to the
  • the tupl* 1-137 SNV ⁇ tupl CTD has been shown to act as a partially dominant gain-of-function mutation as methanol -inducible AOX1 -driven expression of the truncated allele resulted in a significant increase in titer in two different strain backgrounds expressing the wild type TUPl (a GFI2.0 and a complemented GFI5.0) .
  • the TUPl mutation resulted in a truncated allele of Tuplp that encoded amino acids 1-137. Over-expression of this allele already had a dramatic impact (>2x) on mAb titer in a GFI2.0 strain (where N-glycan uniformity is not typically a concern) . Replacement of the 1-137 allele with the wild-type resulted in reduced N-glycan uniformity in GFI5.0 strains. This glycan uniformity phenotype (as well as the mAb titer) was rescued by re-introducing the 1-137 truncated allele into the genome in place of the wild type allele. Therefore, this mutation is broadly applicable to yeast strains and not specific to the strain where the mutation occurred.
  • a polypeptide, such as an immunoglobulin, having a high degree of N-glycan uniformity has greater than about 80% (e.g., 90%) complex glycans, e.g., as measured by ALDI-mass spectroscopic analysis of glycans associated with a polypeptide.
  • the tupl 70 host cells exhibit high polypeptide productivity which refers to any increase in productivity of a heterologous polypeptide in the cell as compared to the levels of expression of the polypeptide in a Tupl wild-type cell.
  • a "heterologous polynucleotide” in a fungal host cell of the present invention in which a tupl TM allele is expressed is a polynucleotide that does not naturally occur in the cell, e.g., because the nucleotide sequence of the polynucleotide does not naturally occur in the fungal cell.
  • a “heterologous polypeptide” is a polypeptide that does not naturally occur in the cell, e.g. , because the amino acid sequence of the polypeptide does not naturally occur in the fungal cell.
  • heterologous polynucleotide encoding a heterologous polypeptide that does not occur naturally in a fungal cell e.g., Pichia cells such as Pichia pastoris
  • an antibody immunoglobulin heavy chain and/or light chain is an antibody immunoglobulin heavy chain and/or light chain.
  • Examples of an antibody containing an immunoglobulin chain which can be encoded by a heterologous polynucleotide in a fungal host cell of the present invention, expressing tupl 0 TM, are
  • heterologous polynucleotides encode: VEGF, HERl, HER2, HER3 , glycoprotein Ilb/IIIa, CD52, IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5 , CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3
  • heterologous polynucleotides encode the light chain or heavy chain immunoglobulin of Abciximab; Adalimumab; Alemtuzumab,- Basiliximab; Bevacizumab; Cetuximab; Certolizumab; Daclizumab;
  • Dalotuzumab Denosumab; Eculizumab; Efalizumab; Gemtuzumab;
  • Ibritumomab tiuxetan,- Infliximab Ibritumomab tiuxetan,- Infliximab; Muromonab-CD3 ; Natalizumab;
  • Omalizumab Palivizumab; Panitumumab; Ranibizumab; Rituximab;
  • Tositumomab or Trastuzuma ; or any immunoglobulin polypeptide containing the light and/or heavy chain variable region or CDRs
  • a "polynucleotide” or “nucleic acid” includes DNA and RNA in single stranded form, double-stranded form or otherwise.
  • the present invention includes isolated polynucleotides encoding the Lupl CTD allele ⁇ e.g., SEQ ID NO: 3 or nucleotides 1-414 thereof).
  • the present invention comprises an isolated polynucleotide that encodes a Pichia pastoris tupl 070 allele; a Saccharo yces cerevisiae t pl CTD allele; a Candida albicans t pl 070 allele or a luyveroiTtyces lactis tupl CTD allele; as well as
  • polynucleotides consisting essentially of such alleles wherein said polynucleotide includes the allele but does not include sequences that are adjacent to such an allele in a host cell chromosome but may be, e.g., operably linked to an expression control sequence such as a promoter.
  • Such polynucleotides including said alleles may be in a vector and/or ectopically maintained in a host cell, e.g., as part of an autonomously replicating unit such as a
  • nucleotide sequence or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a
  • polynucleotide such as DNA or RNA, and means a series of two or more nucleotides.
  • a "coding sequence” or a sequence “encoding” an expression product, such as an RNA or polypeptide is a nucleotide sequence (e.g., heterologous polynucleotide) that, when expressed, results in production of the product (e.g., a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain) .
  • a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain
  • a "cDNA” is a DNA molecule containing the coding sequence of a polynucleotide that encodes a polypeptide which lacks any intron sequences .
  • oligonucleotide refers to a nucleic acid, generally of no more than about 100 nucleotides (e.g., 30, 40, 50, 60, 70, 80, or 90), that may be hybridizable to a
  • Oligonucleotides can be labeled, e.g., by incorporation of 32 P-nucleotides, 3 H-nucleotides, 14 C-nucleotides, 35 S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • “Overexpression” and the like refers to expression of a protein in a cell at levels greater than normal in a wild-type cell.
  • a "protein”, "peptide” or “polypeptide” e.g., a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain ⁇ includes a contiguous string of two or more amino acids.
  • a polypeptide may be indicated with a "p" at the end of a polypeptide name, for example, Tuplp would refer to the Tupl polypeptide.
  • the present invention also encompasses isolated polypeptides encoding the Tupl CTD polypeptide (SEQ ID NO: 10) .
  • a “protein sequence” , “peptide sequence” or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.
  • An isolated polynucleotide or polypeptide will, preferably, be an essentially homogeneous composition of molecules but may contain some heterogeneity.
  • PCR polymerase chain reaction
  • a “promoter” is a DNA regulatory region capable of binding an RNA polymerase in a cell ⁇ e.g. , directly or through other promoter-bound proteins or substances) and initiating
  • a coding sequence ⁇ e.g., of a heterologous polynucleotide, e.g., an immunoglobulin heavy and/or light chain) is "operably linked to", "under the control of”, “functionally associated with” or “operably associated with” a transcriptional or translational control sequence ⁇ e.g., a promoter) when the sequence directs RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
  • a polynucleotide is operably linked to a transcriptional terminator sequence .
  • the present invention includes fungal host cells expressing a tuplTM allele and having a vector which comprises a promoter operably linked to a heterologous polynucleotide (e.g., an
  • the term "vector” includes a vehicle ⁇ e.g., a plasmid) by which a DNA or R A sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.
  • a plasmid is circular, includes an origin (e.g., 2 ⁇ origin) and, preferably includes a selectable marker.
  • yeast markers include URA3 , HIS3, LEU2 , TRP1 and LYS2, which complement specific auxotrophic mutations in a yeast host cell, such as ura3-52, his3-Dl, leu2 ⁇ Dl, trpl-Dl and lys2-201,
  • the plasmid can be maintained in E.coli, it may include a bacterial origin (ori) and/or a selectable market such as the ⁇ -lactamase gene (bla or AMP) .
  • a bacterial origin ori
  • a selectable market such as the ⁇ -lactamase gene (bla or AMP) .
  • Commonly used yeast/B. coli shuttle vectors are the YIp (see Myers efc al., Gene 45: 299-310, (1986)) , YEp (see Myers et al., Gene 45: 299-310, (1986)) , YCp and YRp plasmids.
  • the YIp integrative vectors do not replicate autonomously, but integrate into the host cell genome at low frequencies by homologous recombination.
  • the YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 ⁇ plasmid that serves as an origin of replication (2 ⁇ ori) .
  • the 2 ⁇ ori is responsible for the high copy-number and high frequency of transformation of YEp vectors.
  • the YCp yeast centromere plasmid vectors are autonomously
  • YCp vectors are typically present at very low copy numbers, from 1 to 3 per cell.
  • Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS sequence; but not centromere) that allows the transformed plasmids to be propagated several hundred- fold.
  • YIp, YEp, YCp and YRp are commonly known in the art and widely
  • yeast artificial chromosome is a biological vector. It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication in yeast cells (see Marchuk et al . , Nucleic Acids Res. 16(15): 7743 (1988); Rech et al . , Nucleic Acids Res.
  • Vectors that could be used in this invention include plasmids ⁇ e.g., circular plasmids), viruses, bacteriophage, integratable DNA fragments, and other vehicles that may facilitate introduction of the nucleic acids into the genome of a host cell ⁇ e.g., Pichia pastoris) .
  • Plasmids are the most commonly used form of vector but all other forms of vectors which serve a similar function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al., Cloning Vectors : A Laboratory Manual , 1985 and Supplements, Elsevier, N.Y. , and Rodriguez et al. (eds.),
  • methanol-induction and the like refers to increasing expression of a polynucleotide (e.g., a heterologous polynucleotide e.g. , an immunoglobulin heavy and/or light chain) operably linked to a methanol-inducible promoter.
  • a polynucleotide e.g., a heterologous polynucleotide e.g. , an immunoglobulin heavy and/or light chain
  • AOX1 promoter is an example of a methanol-inducible promoter.
  • the scope of the present invention includes fungal host cells expressing the tupl CTD allele, e.g., comprising a heterologous polynucleotide, encoding a heterologous polypeptide, that is operably linked to a promoter, for example, a methanol-inducible promoter, e.g., AOX1, e.g., in a vector.
  • a promoter for example, a methanol-inducible promoter, e.g., AOX1, e.g., in a vector.
  • Such a method comprises introducing such a methanol-inducible promoter- heterologous polynucleotide construct into such a fungal cell and culturing the host cell in the presence of methanol under
  • the present invention includes fungal host cells expressing the tupl CTD allele for example, wherein the fcupl £7rD allele comprises the nucleotide sequence of SEQ ID NO: 3 or nucleotides 1-414 thereof.
  • the tupl 070 allele is a sequence variant thereof which comprises a nucleotide sequence that differs from but also hybridizes to a complement of a
  • polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or nucleotides 1-414 thereof.
  • the polynucleotides hybridize under low stringency conditions, more preferably under moderate stringency conditions and most preferably under high stringency conditions.
  • a polynucleotide is "hybridizable" to another polynucleotide when a single stranded form of the nucleic acid molecule (e.g. , either strand) can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook, et al . , supra) . The conditions of temperature and ionic strength determine the
  • Low stringency hybridization conditions may be 55°C, 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5X SSC, 0.5% SDS.
  • Moderate stringency hybridization conditions are similar to the low stringency
  • High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50% formamide, 5X or 6X SSC and, optionally, at a higher temperature (e.g., 57°C, 59°C, 60°C, 62°C, 63°C, 65°C or 68°C) .
  • SSC is 0.15M NaCl and 0.015M sodium citrate.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
  • the tupl CTD allele comprises a nucleotide sequence which is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95%
  • the Tupl 0 TM polypeptide comprises an amino acid sequence which is at least about 70% similar or identical, preferably at least about 80% similar or identical, more preferably at least about 90% similar or identical and most preferably at least about 95% similar or identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence set forth in SEQ ID NO: 10 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences; but which retains the ability to form a complex with SSN6/CYC8; and/or cause high polypeptide (e.g., immunoglobulin) productivity in a fungal host cell which expressed the protein; and/or cause high N-glycan uniformity of polypeptides (e.g., immunoglobulins) expressed in a fungal host cell having the polypeptides; but which protein lacks at least the C-terminal half of
  • Sequence identity refers to exact matches between the
  • BLAST ALGORITHMS Altschul, S.F., et al., J. Mol. Biol. (1990) 215:403-410; Gish, W. , et al., Nature Genet. (1993) 3:266-272; Madden, T.L., et al . , Meth. Enzyraol .
  • the present invention encompasses isolated fungal host cells ⁇ e.g. , Pichia cells such as Pichia pastoris) that express Tupl CTD (e.g., from Pichia pastoris, Saccharomyces cerevisiae or Candida albicans) , e.g., including a polynucleotide encoding a heterologous polypeptide (e.g., an immunoglobulin chain).
  • the heterologous polypeptide is encoded by a heterologous polynucleotide operably linked to a promoter, e.g., a methanol -inducible promoter.
  • the fungal host cells of the present invention are made by mutating endogenous chromosomal Tupl in an isolated Pichia pastoris cell to a tupl 0 TM allele, for example, wherein said mutated rupl encodes a polypeptide comprising amino acids 1-137 of Pichia pastoris Tupl; or, by introducing a polynucleotide that encodes Tupl CTD into a fungal host cell (e.g., that comprises wild-type Tupl or another Tupl mutated allele, e.g., a null allele) .
  • a polynucleotide that encodes Tupl CTD e.g., that comprises wild-type Tupl or another Tupl mutated allele, e.g., a null allele
  • Fungal host cells of the present invention may be genetically engineered so as to express particular glycosylation patterns on polypeptides (e.g., heterologous polypeptides such as
  • the fungal host cell is a yeast cell, such as a methylotrophic yeast cell, which, for example, is selected from the group consisting of any Pichia cell, Pichia pastoris, Pichia flnlandica, Pichia
  • Pichia koclamae Pichia membranaefaciens
  • Pichia minuta Ogataea minuta, Pichia lindneri
  • Pichia opuntiae Pichia thermotolerans
  • Pichia salictaria Pichia guercuum
  • Pichia pijperi Pichia stiptis
  • Pichia ethanolica Pichia, Saccharomyces
  • N-glycan and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide.
  • N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • Predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N- acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)).
  • GalNAc N-acetylgalactosamine
  • GlcNAc N-acetylglucosamine
  • sialic acid e.g., N-acetyl-neuraminic acid (NANA)
  • N-glycans have a common pentasaccharide core of Man 3 GlcNAc 2 ("Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine) .
  • N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars ⁇ e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man 3 GlcNAc 2 (“Man 3 ") core structure which is also referred to as the "trimannose core", the "pentasaccharide core” or the "paucimannose core”.
  • N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid) .
  • a "high mannose” type N-glycan has five or more mannose residues.
  • a "complex” type N-glycan typically has 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 "trimannose" core.
  • Complex N-glycans may also have galactose (“Gal”) or N- acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl) .
  • Gal galactose
  • GalNAc N- acetylgalactosamine residues
  • sialic acid or derivatives e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl
  • Complex N-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc and core fucose (“Fuc”) .
  • Complex N-glycans may also have multiple antennae on the "trimannose core, " often referred to as "multiple antennary g
  • a “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3-mannose arm of the trimannose core and zero or more mannoses on the 1,6-mannose arm of the trimannose core.
  • Hybrid N-glycans may also have a galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residue that are optionally modified with sialic acid or derivatives (e.g., "NANA” or "NeuAc”) attached to the GlcNAc on the 1,3-mannose arm.
  • the various N-glycans are also referred to as "glycoforms .
  • PNGase or “glycanase” or “glucosidase” refer to peptide N- glycosidase F (EC 3.2.2.18).
  • the isolated fungal host cell is glycoengineered .
  • such a cell has been genetically engineered to produce glycoproteins where the N- or O-linked glycosylation are modified from their native form, e.g., either through inactivation or deletion of genes involved in N-glycosylation such as OCH1, ALG3 , PNOl, and/or BMT2 or genes involved in O-glycosylation such as PMTl, PMT2 and/or PMT4 or though heterologous expression of glycosyltransferases such as GnTI, GnTII, GalT, and/or SialT, or mannosidases such as MNSI and/or MNSII.
  • a glycoengineered isolated fungal host cell comprises any one or more of the following characteristics:
  • an isolated Tupl cro fungal host cell such as a Pichia cell ⁇ e.g., Pichia pastoris
  • a nucleic acid that encodes an cc- 1, 2-mannosidase that has a signal peptide that directs it for secretion.
  • the T pl 010 host cell is engineered to express an exogenous oc-1,2- mannosidase enzyme having an optimal pH between 5.1 and 8.0, preferably between 5.9 and 7.5.
  • the exogenous enzyme is targeted to the endoplasmic reticulum or Golgi apparatus of the host cell, where it trims N-glycans such as Man 8 GlcNAc 2 to yield Man 5 GlcNAc 2 .
  • N-glycans such as Man 8 GlcNAc 2 to yield Man 5 GlcNAc 2 .
  • the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a
  • polynucleotide encoding the heterologous polypeptide (s) into such a Tupl 010 , a-1, 2-mannosidase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, ( ii) isolating the heterologous polypeptide (s) from the host cell.
  • the invention also encompasses a method for producing a heterologous recombinant glycoprotein comprising an N-glycan structure that comprises a Man 5 GlcNAc 2 glycoform in a Tupl OTD fungal host cell that does not display alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the method comprising the step of introducing into the Tupl 070 fungal host cell, a polynucleotide encoding the heterologous recombinant glycoprotein, and a
  • polynucleotide encoding an alpha- 1,2 mannosidase enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising: (a) an alpha- 1,2 mannosidase catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0; fused to (b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the mannosidase enzyme to the ER or Golgi apparatus of the host cell; and culturing the fungal host cell under conditions favorable to expression of the heterologous recombinant glycoprotein, whereby, upon expression and passage of the heterologous recombinant glycoprotein through the ER or Golgi apparatus of the host cell, in excess of 30 mole % of the N-glycan structures attached thereto have a Man 5 GlcNAc 2 glycoform that can serve as a substrate for GlcNAc transferase I in vivo.
  • Isolated Tupl CTD fungal host cells of the present invention such as Pichia host cells (e.g., Pichia pastoris) are, in an embodiment of the invention, genetically engineered to eliminate glycoproteins having alpha-mannosidase-resistant N-glycans by deleting or disrupting one or more of the ⁇ -mannosyltransferase genes (e.g., BMTl, BMT2 , BMT3 , and/or BMT4 ) (See, U.S. Patent No. 7,465,577) or abrogating translation of RNAs encoding one or more of the beta-mannosyltransferases using interfering RNA, antisense RNA, or the like.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising ⁇ i) introducing a polynucleotide encoding the
  • ⁇ - mannosyltransferase ⁇ e.g., bmtl ⁇ , bmt2 ⁇ , bmt3 ⁇ , and/or bmt4 ⁇
  • Isolated Tupl 070 fungal host cells e.g., Pichia, e.g., Pichia pastoris
  • Isolated Tupl 070 fungal host cells also include those that are genetically engineered to eliminate glycoproteins having
  • phosphomannose residues e.g., by deleting or disrupting one or both of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos . 7,198,921 and 7,259,007), which can include deleting or disrupting one or more of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos . 7,198,921 and 7,259,007), which can include deleting or disrupting one or more of the
  • such fungal host cells produce glycoproteins that have predominantly an N-glycan selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans wherein complex N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 3 GlcNAc 2 , GlcNACu- 4 )Man 3 GlcNAc 2 , NANA( 1 -4)GlcNAC(i-4)Man 3 GlcNAC 2 , and NANA ( i -4 )Gal (1 _
  • hybrid N-glycans are, in an embodiment of the invention, selected from the group consisting of an 5 GlcNAc 2 ,
  • NANAGalGlcNAcMan 5 GlcNAc 2 ; and high mannose N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 6 GlcNAc 2 , Man 7 GlcNAc 2 , Mang 8 lcNAc 2 , and Man 9 GlcNAc 2 .
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a
  • polynucleotide encoding the heterologous polypeptide (s) into such a Tupl , phosphomannosyl transferase " ⁇ e.g. , pnol " and/or mnn4b ⁇ ) host cell and (ii) culturing the host cell under conditions
  • heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous
  • polypeptide (s) from the host cell is polypeptide (s) from the host cell.
  • Isolated Tupl CTD fungal host cells such as Pichia host cells ⁇ e.g., Pichia. pastoris
  • Isolated Tupl CTD fungal host cells include those that are genetically engineered to include a nucleic acid that encodes the Leishmania sp. single-subunit oligosaccharyltransferase STT3A protein, STT3B protein, STT3C protein, STT3D protein, or
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide (s) into such a Tu lTM, ⁇ Leishmania STT3A*, Leishmania STT3B * , Leishmania STT3C*, and/or Leishmania STT3D * ) host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • Isolated Tupl CTD fungal host cells e.g., Pichia pastoris
  • Isolated Tupl CTD fungal host cells also include those that are genetically engineered to eliminate nucleic acids encoding dolichol-P- an dependent alpha(l-3) mannosyltransferase, e.g., Alg3 , such as described in U.S. Patent Publication No. US2005/0170452.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a
  • heterologous polypeptide (s) into such a Tupl 070 , Alg3 ⁇ host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous
  • polypeptide (s) in the cell and, optionally, ⁇ iii) isolating the heterologous polypeptide (s) from the host cell.
  • Isolated Tupl CTD fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris) expressing a
  • polypeptide having an endomannosidase activity ⁇ e.g., human ⁇ e.g., human liver
  • rat or mouse endomanosidase that is targeted to a vesicular compartment within the host cell.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous
  • polypeptide (s) into such a TplTM 3 , endomannosidase "1" host cell and (ii) culturing the host cell under conditions favorable to
  • heterologous polypeptide (s) expression of the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • Isolated Tupl 070 fungal host cells such as Pichia cells [e.g., Pichia pastoris) of the present invention are, in an embodiment of the invention, engineered for producing a recombinant sialylated glycoprotein in the host cell, e.g., wherein the host cell is selected or engineered to produce recombinant glycoproteins
  • polynucleotides encoding a bifunctional UDP-N-acetylglucosamine-2 - epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9- phosphate synthase, and a CMP- sialic acid synthase,- (b)
  • transforming into the host cell a polynucleotide encoding a CMP- sialic acid transporter; and (c) transforming into the host cell a polynucleotide molecule encoding a 2 , 6-sialyltransferase catalytic domain fused to a cellular targeting signal peptide, e.g., encoded by nucleotides 1-108 of the S.
  • a recombinant sialylated glycoprotein comprising a glycoform selected from the group consisting of NANA (1-4 )Gal a- 4 )GlcNAC(i- 4 )Man 3 Glc Ac 2 glycoform is produced.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a
  • heterologous polypeptide (s) into such a czrlTM ⁇ 111 , bifunctional UDP-N-acetylglucosamine-2-epimerase/N- acetylmannosamine kinase * , N-acetylneuraminate- 9-phosphate
  • isolated czrl mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris)
  • Pichia cells e.g., Pichia pastoris
  • isolated czrl mutant fungal host cells of the present invention are, in an embodiment of the invention, engineered for generating galactosylated proteins, e.g., having a terminal galactose residue and essentially lacking fucose and sialic acid residues on the glycoprotein.
  • the isolated czrl mutant fungal host cell comprises an isolated nucleic acid molecule encoding ⁇ -galactosyltransferase activity and at least a polynucleotide encoding UDP-galactose transport activity, UDP-galactose C4 epimerase activity, galactokinase activity or galactose-1-phosphate uridyl transferase, e.g., wherein the host cell is genetically engineered to produce N-linked oligosaccharides having terminal GlcNAc residues and comprising a polynucleotide encoding a fusion protein that in the host cell transfers a galactose residue from UDP-galactose onto a terminal GlcNAc residue of an N-linked oligosaccharide branch of an N-glycan of a
  • glycoprotein wherein the N-linked oligosaccharide branch is selected from the group consisting of GlcNAcpl, 2-Manal; GlcNAcpl,4- Man l,3, GlcNAcpl, 2 -Manal , 6 , GlcNAcPl , 4-Manal , 6 and GlcNAc l , 6- anal,6; wherein the host cell is diminished or depleted in dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl a-1, 3 mannosyltransferase activity, and wherein the host cell produces a glycoprotein having one or more galactose residues.
  • the scope of the present invention includes methods for producing one or more heterologous
  • polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide (s) into such a host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • an isolated czrl mutan fungal host cell of the present invention such as Pichia cells ⁇ e.g., Pichia pastoris
  • the scope of the present invention includes methods for producing one or more heterologous
  • polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide (s) into such a Tupl CTD / ochl ' host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, ⁇ iii) isolating the heterologous polypeptide (s) from the host cell .
  • Isolated Tupl 0 TM fungal host cells of the present invention such as Pichia cells ⁇ e.g., Pichia pastoris) expressing a
  • galactosyltransferase e.g., an alpha 1, 3 -galactosyltransferase or a beta 1,4- galactosyltransferase are part of the present
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous
  • Isolated Tupl 0 TM fungal host cells of the present invention such as Pichia cells ⁇ e.g., Pichia pastoris) expressing a
  • nucleotide sugar transporter are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide (s) into such a Tupl 070 , nucleotide sugar transporter" host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • Isolated Tupl fungal host cells of the present invention such as Pichia cells ⁇ e.g. , Pichia pastoris) expressing a
  • sialyltransferase are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a
  • polynucleotide encoding the heterologous polypeptide (s) into such a rupl crD , sialyltransferase* host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • Isolated Tupl CTD fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris) expressing an
  • acetylglucosaminyl transferase e.g., GNTl or GNT2 or GNT4 are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous
  • polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide (s) into such a Tupl 070 ,
  • heterologous polypeptide (s) in the cell and, optionally, (iii) isolating the heterologous polypeptide (s) from the host cell.
  • Fungal host cells ⁇ e.g., Pichia
  • Fungal host cells also include those that are genetically engineered to eliminate glycoproteins having
  • phosphomannose residues e.g., by deleting or disrupting one or both of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos. 7,198,921 and 7,259,007), which can include deleting or disrupting the MNN4A gene or abrogating translation of RNAs encoding one or more of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos. 7,198,921 and 7,259,007), which can include deleting or disrupting the MNN4A gene or abrogating translation of RNAs encoding one or more of the
  • a fungal host cell has been genetically modified to produce glycoproteins that have predominantly an N-glycan selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans wherein complex N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 3 GlcNAc 2 , GlcNAC ( i-
  • hybrid N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 5 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 , and
  • NANAGalGlcNAcMan 5 GlcNAc 2 ,- and high mannose N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 6 GlcNAc 2 , Man 7 GlcNAc 2 , Mang 8 lcNAc 2 , and Man 9 GlcNAc 2 ⁇
  • the scope of the present invention includes such an isolated fungal host cell (e.g., Pichia) expressing a tupl 0 TM allele and including a
  • polynucleotide encoding a heterologous polypeptide (e.g. , an immunoglobulin chain) .
  • a heterologous polypeptide e.g. , an immunoglobulin chain
  • the term "essentially free of” as it relates to lack of a particular sugar residue, such as fucose, or galactose or the like, on a glycoprotein is used to indicate that the glycoprotein composition is substantially devoid of N-glycans which contain such residues.
  • essentially free means that the amount of N-glycan structures containing such sugar residues does not exceed 10%, and preferably is below 5%, more preferably below 1%, most preferably below 0.5%, wherein the percentages are by weight or by mole percent .
  • glycoprotein composition "lacks" or “is lacking” a particular sugar residue, such as fucose or galactose, when no detectable amount of such sugar residue is present on the N-glycan structures.
  • glycoprotein compositions are expressed, as discussed herein, and will "lack fucose," because the cells do not have the enzymes needed to produce fucosylated N-glycan structures.
  • a composition may be "essentially free of fucose” even if the composition at one time contained fucosylated N-glycan structures or contains limited, but detectable amounts of fucosylated N-glycan structures as described above.
  • the present invention includes isolated polynucleotides comprising mutated alleles of Tupl from any fungal cell, e.g.
  • Pichia such as Pichia pastoris, Saccharomyces cerevisiae or Candida albicans.
  • the mutated alleles of Tupl comprise a C- terminal deletion (tupl CTD ) .
  • the Pichia pastoris tupl CTD encodes amino acids about 1 to about 137 (e.g., of SEQ ID NO: 2)
  • the Saccharo yces cerevisiae tupl CTD encodes amino acids about 1 to about 139 (e.g., of SEQ ID NO: 5)
  • the Candida albicans tupl CTD encodes amino acids about 1 to about 120 ⁇ e.g.
  • the Pichia pastoris tupl 0 TM allele comprises nucleotides 1-414 SEQ ID NO: 3; the Saccharomyces cerevisiae tupl CTD allele comprises nucleotides 1-402 of SEQ ID NO: 4; and/or the Candida albicans tupl CTD allele comprises nucleotides 1-360 of SEQ ID NO: 6.
  • the present invention further encompasses vectors (e.g., plasmids) comprising polynucleotides that comprise a tu l 0 TM allele; as well as isolated polypeptides encoding Tu l" 0 .
  • vectors e.g., plasmids
  • a Tupl CTD protein retains the ability to form a complex with SSN6/CYC8; and/or causes a phenotype wherein growth is resistant to Ca 2+ (e.g., CaCl 2 , e.g., 0.2M CaCl 2 ) for example, when grown on solid growth medium such as on a plate ; and/or cause high polypeptide (e.g., immunoglobulin) productivity in a fungal host cell which expressed the protein and/or cause high N-glycan uniformity of polypeptides (e.g., immunoglobulins) expressed in a fungal host cell having the polypeptides; but which protein lacks at least the C-terminal half of the wild-type polypeptide, for example, which lacks C-terminal WD40 repeats.
  • Ca 2+ e.g., CaCl 2 , e.g., 0.2M CaCl 2
  • the Pichia pastoris TUP1 cDNA sequence comprises the nucleotide sequence:
  • the Pichia pastoris Tupl protein comprises the amino acid sequence:
  • the Pichia pastoris tupl 0 TM allele cDNA comprises the following nucleotide sequence wherein the allele differs from wild-type Tpl at the indicated uppercase underscored "T" nucleotide (this allele may also be referred to as Q138stop) .
  • This mutation introduces a missense mutation:
  • the Pichia pastoris tupl 010 allele encodes a polypeptide comprising the following amino acid sequence : MSYNRPLPNTTSVANQQSRQRLEDLLQGIKKEFENLSSETSFY LHQDEVEMKFTQQNRELQNIRNA VYELDVAHRKM DAYE EILRYKQELE RDRLLQQQQQQQHQPQHQQPGLENRDSSAYNQQLPPPNL NAH (SEQ ID NO: 10)
  • Saccharomyces In an embodiment of the invention, the Saccharomyces
  • Tupl cDNA sequence comprises the nucleotide sequence: ATGACTGCCAGCGTTTCGAATACGCAGAATAAGCTGAATGAGCTTCTCGATGCCATCAGA CAGGAGTTTCTCCAAGTCTCACAAGAGGCAAATACCTACCGTCTTCAAAACCAAAAGGAT TACGATTTCAAAATGAACCAGCAGCTGGCTGAGATGCAGCAGATAAGAAACACCGTCTAC GAACTGGAACTAACTCACAGGAAAATGAAGGACGCGTACGAAGAAGAGATCAAGCACTTG AAACTAGGGCTGGAGCAAAGAGACCATCAAATTGCATCTTTGACCGTCCAGCAACAGCGG CAACAGCAACAGCAGCAACAGGTCCAGCAGCATTTACAACAACAGCAACAGCAGCAGCTAGCC GCTGCATCTGCATCTGTTCCAGTTGCGCAACAACCACCGGCTACTACTTCGGCCACCGCC ACTCCAGCAGCAAACACAACTGGTTCGCCATCGGCCTTCCCAGTACAAGCTAGCCGT CCTAA
  • cerevisiae Tupl protein comprises the amino acid sequence:
  • Candida albicans Tupl cDNA sequence comprises the nucleotide sequence:
  • Kluyveromyces lactis Tupl cDNA sequence comprises the nucleotide sequence:
  • the present invention encompasses methods for making a polypeptide (e.g., an immunoglobulin heavy and/or light chain or an antibody or antigen-binding fragment thereof) comprising
  • an isolated fungal host cell of the present invention e.g., Pichi , e.g., Pichia pastoris
  • a heterologous polynucleotide e.g., operably linked to a promoter (e.g., a methanol -inducible
  • a liquid culture medium e.g., YPD medium (e.g. , comprising 1% yeast extract, 2% peptone, 2% glucose)
  • a liquid culture medium e.g., YPD medium (e.g. , comprising 1% yeast extract, 2% peptone, 2% glucose)
  • methanol e.g., 1% yeast extract, 2% peptone, 2% glucose
  • Expression of the polynucleotide may be induced when the promoter is methanol-inducible and the host cells are grown in the presence of methanol .
  • An expression system comprising the fungal host cells of the present invention (e.g., tupl CTD ) comprising the promoter, operably linked to the heterologous polynucleotide, e.g., in an ectopic vector or integrated into the genomic DNA of the host cell, forms part of the present invention.
  • a composition comprising the fungal host cell which includes the promoter operably linked to the heterologous polynucleotide in liquid culture medium also forms part of the present invention.
  • a method for expressing a heterologous polypeptide does not comprising starving the fungal host cells of a nutrient such as a carbon source such as glycerol or glucose.
  • a nutrient such as a carbon source such as glycerol or glucose.
  • Other embodiments of the present invention include methods wherein the cells are starved.
  • the present invention comprises methods for expressing a polypeptide in a fungal glycosylation mutant host cell, e.g., as discussed herein, wherein the host cell comprises a promoter (e.g., methanol -inducible) operably linked to a promoter (e.g., methanol -inducible) operably linked to a promoter (e.g., methanol -inducible) operably linked to a promoter (e.g., methanol -inducible) operably linked to a promoter (e.g., methanol -inducible) operably linked to a promoter
  • heterologous polynucleotide encoding the polypeptide wherein the host cell is or is not starved and is cultured in the presence of methanol .
  • the heterologous amino acids in an embodiment of the invention, the heterologous amino acids
  • the polynucleotide that is operably linked to the promoter is in a vector that comprises a selectable marker.
  • the fungal host cells e.g., tupl CTO
  • the selectable marker is a drug resistance gene, such as the zeocin resistance gene, and the cells are grown in the presence of the drug, such as zeocin.
  • heterologous polypeptide expression using a methanol -inducible promoter includes three phases, the glycerol batch phase, the glycerol fed-batch phase and the methanol fed-batch phase.
  • the glycerol batch phase (GBP)
  • fungal host cells e.g., tupl CTD
  • GFP glycerol fed- batch phase
  • a limited glycerol feed is initiated following exhaustion of the glycerol in the previous phase, and cell mass is increased to a desired level prior to methanol -induction.
  • the third phase is the methanol fed-batch phase (MFP) , in which methanol is fed at a limited feed rate or maintained at some level to induce the methanol- inducible promoters for protein expression.
  • MFP methanol fed-batch phase
  • a limited glycerol feed can be simultaneously performed for promoting production when necessary.
  • the present invention encompasses methods for making a heterologous polypeptide (e.g., an immunoglobulin) comprising introducing, into an isolated fungal host cell, for example, LuplTM ⁇ e.g., Pichia, such as Pichia pastoris) a
  • heterologous polynucleotide encoding said polypeptide that is operably linked to a methanol-inducible promoter of the present invention and culturing the host cells
  • a batch phase ⁇ e.g., a glycerol batch phase
  • a non-fermentable carbon source such as glycerol
  • a batch-fed phase ⁇ e.g. , a glycerol batch-fed phase
  • additional non-fermentable carbon source ⁇ e.g., glycerol
  • an initial seed culture is grown to a high density ⁇ e.g., OD eoo of about 2 or higher) and the fungal host cells grown in the seed culture are used to inoculate the initial batch phase culture medium .
  • the fungal host cells are grown in a transitional phase wherein cells are grown in the presence of about 2 ml methanol per liter of culture.
  • the cells can be grown in the transitional phase until the methanol concentration reaches about zero.
  • the fungal host cells e.g., Pichia. cells such as Pichia pastoris
  • the fungal host cells are grown under any 1, 2, 3, 4, 5 or 6 of the following conditions:
  • trace minerals/nutrients such as copper, iodine, manganese, molybdenum, boron, cobalt, zinc, iron, biotin and/or sulfur, e.g., CuS0 4 , Nal, MnS0 4 , Na 2 Mo0 , H 3 BO 3 , CoCl 2 , ZnCl , FeS0 4 , biotin and/or H 2 S0 4 ; and/or
  • an anti-foaming agent e.g., silicone
  • an oxygen concentration of about 20% saturation or higher
  • the present invention provides methods for making polypeptides, such as immunoglobulin chains, antibodies or antigen-binding fragments thereof having modified glycosylation patterns, for example, by expressing a polypeptide in a fungal host cell that introduces a given glycosylation pattern and/or by growing the fungal host cell under conditions wherein the glycosylation is introduced. Some of such host cells are discussed herein.
  • the invention provides methods for making a heterologous protein that is a glycoprotein comprising an N-glycan structure that comprises a Man 5 GlcNAc 2 glycoform; comprising introducing a polynucleotide encoding the polypeptide wherein the polynucleotide is operably linked to a promoter of the present invention into a host cell and culturing the host cell under conditions wherein the polypeptide is expressed with the Man 5 GlcNAc 2 glycoform and/or lacking fucose.
  • the present invention is intended to exemplify the present invention and not to be a limitation thereof.
  • the methods and compositions disclosed below fall within the scope of the present invention
  • Example 1 Identification and annotation of the complete genome sequence of P. pastoris strain RRL-y11430
  • Biosciences (Beverly, MA) yielding 9,411,042 bases on 4 large contigs and one smaller contig of 34,728 bp (nucleotide base pairs) that could not be resolved, consistent with the previously
  • Example 2 Next Generation Sequencing of Glycoengineered P. pastoris strains and Genome-scale Single Nucleotide Variation - Identification of a mutation in P. pastoris TUP1
  • Genomic DNA was isolated for the wild type P. pastoris strain NRRL-yll430, as well as glycoengineered strains YGLY16-3, YGLY24-3, YGLY1703, YGLY3853, YGLY4754, YGLY8316, YGLY8323, and YGLY8813, by standard means (Piper, 1996) .
  • DNA was quantified using a standard spectrophotometer (Eppendorf, Regensberg, Germany) and a
  • SNV single nucleotide variations
  • maq.pl a utility program in the MAQ package, to call SNVs by the following commands : maq.pl easyrun -a 250 -e 3 -q 40 -m 2 -D 256 -E 20 -N 2 -w 5 -b 60 -B 2
  • Genomic DNA was isolated from strains NRRL-yll430, YGLY16-3 YGLY3853, YGLY4754, YGLY4799, YGLY6903, YGLY8292, YGLY8316,
  • YGLY8323, YGLY12501, and YGLY13992 ⁇ see Figure 2) by the following procedure: a smear of yeast cells ( ⁇ 10 s ) was combined in a 1.5ml centrifuge tube with ⁇ 30 ⁇ 1 of 0.5mm glass beads, 50 ⁇ 1 of
  • Phenol/Chloroform (Sigma, St. Louis, MO), and 150 ⁇ 1 of lysis buffer (1% SDS, 2% TritonX-100, lOOmM NaCl, lmM EDTA, lOmM Tris) ; the mixture vortexed for 20 seconds, centrifuged for 10 minutes at 15000 RPM, the aqueous layer removed and combined with ⁇ 50 ⁇ 1 of 100% ethanol, mixed and centrifuged for 20 minutes at 15000 RPM, the supernatant removed, the DNA pellet washed and dried, then resuspended in 20 ⁇ 1 of lOmM Tris. This DNA was used as a template for a PGR reaction using primers RCD909 (5'-
  • CCACAATGCTACTACAACACTCTTCCTG-3 ' CCACAATGCTACTACAACACTCTTCCTG-3 '
  • CD910 5 1 - CGACACTGAGAAGATAAGGAGTGAGG-3 '
  • This PCR product was gel isolated and used as a template for a Sanger DNA sequencing reaction using primer RCD911 (5 1 - CCCAAATGTCGTACAACAGACCATTGCC-3 ' located at the TUP1 ATG start site.
  • strains YGLY8316 and YGLY8323 had a mutant tupl-l allele with nucleotide 412 changed from C to T ⁇ see Figure 3) , thus resulting in a change at codon 138 from CAT (glutamine) to TAG (stop) .
  • This nonsense mutation results in an ORF that encodes a truncated protein expressing only the first 137 amino acids of the Tupl protein. Based on comparison with S. cerevisiae Tuplp, this results in a protein that contains the N- terminal CYC8 interaction domain and part of the first repression domain but lacks the complete C-terminal repression domain. Similar fragments expressed in S.
  • Tupl variants that could exert partial repression activity at some loci but seemingly lost all repression activity at other loci (Zhang, 2002) .
  • the Tuplp expressed in these strains has either lost some or all of the repression function at certain key loci or acts as a dominant negative allele to remove repression exerted by CYC8 through this remaining interaction domain.
  • Parental strain NRRL-yll430 was confirmed to have a CAG at codon 138 in agreement with the whole genome Sanger sequencing (Example 1) .
  • Strains YGLY16-3, YGLY3853, and YGLY8292 were shown to be wild type (CAG), whereas strains YGLY4754, YGLY4799, YGLY6903, YGLY12501 and YGLY13992 were shown to be mutant (TAG) , indicating that the mutation occurred at the strain construction step from YGLY3853 to YGLY4754 (see Figure 2) .
  • the sequencing data are shown for strains YGLY8323, YGLY12501, and YGLY8292 to illustrated the location of the mutation ( Figure 3). Subsequent
  • PCR/sequencing genotyping reactions also paired primer RCD911 with primer RCD921 (5 ' -CTGTAGGCGAAGTTTTAGCAATGGCCG-3 1 ) , which generate a 1.1Kb PCR product, and then using the same RCD911 sequencing primer.
  • RCD911 primer to complement the tupl-1 allele to wild type
  • the P. pastoris wild type TUP1 gene including 500bp of the promoter region and 200bp of the terminator region, was PCR amplified from NRRL-yll430 genomic D A using primers RCD916 5'- GCGGCCGC CACAATGCTACTACAACACTCTTCCTG- 3 ' and RCD917 5'- CCATGGCGACACTGAGAAGATAAGGAGTGAGG- 3 ' and the resulting gel
  • Example 5 Generation of an anti-HER2 monoclonal antibody ⁇ mAb) expressing glycoengineered Pichia pastoris strain
  • Plasmid pGLY5883 was generated by fusing DNA sequences encoding the ⁇ and ⁇ chains of the Trastuzumab anti-HER2 monoclonal antibody (Carter, 1992 ⁇ individually to the P. pastoris AOX1 promoter and is depicted in Figure 6.
  • DNA of this plasmid was digested with Spel to linearize and transformed by standard electroporation method (Pichia kit, Invitrogen, Carlsbad, CA) into the P. pastoris glycoengineered strain YGLY8316 ( Figure 2) , which has been modified to produce complex-type human N-glycans with terminal ⁇ -l , 4-galactose (GFI5.0, Figure 7; Davidson U.S. Patent no. 7,795,002).
  • Clones were selected on medium containing Zeocin and further screened by standard cultivation in 96 deep well plates and 0.5L Sixfors multifermentation fermenters (ATR Biotech, Laurel, MD; Barnard, 2010) .
  • One positive expression clone was saved and named YGLY13992.
  • This P. pastoris mAb-secreting strain was further modified by selection of clones on medium containing 1 g/L 5- flouro-orotic acid (5-FOA) to evict the URA5 gene.
  • 5- flouro-orotic acid (5-FOA) was selected from medium containing 1 g/L 5- flouro-orotic acid (5-FOA) to evict the URA5 gene.
  • 5- flouro-orotic acid 5-FOA
  • One confirmed ura5 auxotrophic clone was saved and named YGLY16656.
  • Example 6 Complementation of the tupl-1 allele to wild type in a P. pastoris mAb-producing strain Plasmid pGLY5640 was digested with Hpal to linearize and transformed into the P. pastoris glycoengineered strain YGLY16656, selecting for transformants on medium lacking uracil. Strain
  • YGLY16656 is a ura5 mutated descendent of strain YGLY13992, which is glycoengineered to produce secreted proteins with human N- glycans with terminal ⁇ -1, 4 -galactose (GFI5.0, Figure 7; Davidson U.S. Patent no. 7,795,002), and expresses the secreted Trastuzumab anti-HER2 mAb under control of the strong methanol -inducible AOXl promoter ⁇ See strain lineage tree, Figure 2) . Transformants were screened by colony PCR and sequencing with primer RCD911 as shown in Example 3 to confirm integration of the plasmid.
  • TUPl/tupl-1 as shown in the cartoon in Figure 8.
  • the isolated 5- FOA resistant clones were screened by colony PCR and sequencing with primer RCD911 as in Example 3.
  • One clone confirmed to contain only the wild type TUPl gene by presence of only a C at nucleotide 412 of the TUPl ORF was saved and named YGLY19193.
  • Plasmid pGLY579 ( Figure 9) , which contains the URA5 gene and HIS3 localization sequence, was digested with Sfil to linearize and transformed into strain YGLY19193 to complement the URA5 auxotrophy.
  • Clones were selected on medium lacking uracil and two strains, confirmed to still contain only the TUPl wild type allele by PCR/sequencing (as in example 3) were saved and named YGLY19250 and YGLY19251 ( Figure 8) .
  • Example 7 The tupl-1 mutation increases yield of secreted antibody under mini-bioreactor conditions
  • TUPl wild type complemented, anti-HER2 mAb expressing, glycoengineered P. pastoris strains YGLY19250 and YGLY19251
  • Seed cultures were prepared by inoculating strains from YSD plates to a Whatman 24-well Uniplate (10 ml, natural polypropylene) containing 3.5 ml of 4% BMGY medium (Invitrogen, Carlsbad, CA) buffered to pH 6.0 with potassium phosphate buffer. The seed cultures were grown for approximately 65-72 hours in a temperature controlled shaker at 24 °C and 650 rpm agitation.
  • Microaerobicl mode and the fermentations were controlled at 200% dissolved oxygen, pH at 6.5, temperature at 24°C and agitation at 800rpm.
  • the induction phase was initiated upon observance of a dissolved oxygen (DO) spike after the growth phase by adding bolus shots of methanol feed solution (100% [w/w] methanol, 5 mg/1 biotin and 12.5 ml/1 PTM2 salts), 50 ⁇ 1 in the morning and 125 ⁇ 1 in the afternoon.
  • DO dissolved oxygen
  • the cell-free culture supernatant was harvested by centrifugation at 2500 x g in a Beckman swinging bucket centrifuge and subjected to protein A purification by standard methods (Jiang, 2011) .
  • Antibody was quantified by reverse phase HPLC and calculated on a per liter basis. Strains were each cultured in duplicate. The YGLY13992 (tupl-l) parental control strain produced 535 +/- 28 mg/L of purified secreted anti-HER2 mAb, while the YGLY19250 and YGLY19251 (TUP1) wild type complemented strains produced on average 104 +/- 16 mg/L, or 5.1 fold less antibody than the parental control
  • Protein Express 200 method and displayed in SDS-PAGE graphical format ( Figure 10B) .
  • Antibody produced by TUPl and tupl-l strains is similar in quality and folding as shown by non-reducing Labchip analysis, but the tupl-l strain yields significantly higher titer.
  • Example 8 Expression of an antibody in a glycoengineered, strain with a tupl-l mutation results in increased N-glycan uniformi y
  • Purified antibody from the YGLY13992 (tupl-1) anti-HER2 antibody-expressing strain and the complemented strains YGLY19250 and YGLY19251 was further analyzed by enzymatic deglycosylation with PNGaseF (New England Biolabs, Ipswich, MA) and MALDI-TOF mass spectrometry as described previously (Choi, 2003).
  • PNGaseF New England Biolabs, Ipswich, MA
  • MALDI-TOF mass spectrometry
  • DmMNSII melanogaster Mannosidase II
  • hGnTII Rattus norvegicus GlcNAc Transferase II
  • Plasmid pGLY167b is marked with the P. pastoris HIS1 gene (complementing the hisl auxotrophy of YGLY3853) and integrates into the P.
  • YGLY4754 was then further streak isolated to yield strain YGLY20599 (argl, tupl-1) .
  • a TUPl wild type sister clone of YGLY4754 was identified, that also contained the pGLY167b plasmid, properly localized, named YGLY4829 (argl, TUP1; see Figure 2) .
  • plasmid pGLY5883 was inserted into plasmid pGLY5883 as a Notl/Ascl fragment, resulting in a plasmid encoding the ⁇ and ⁇ chains of the Trastuzumab anti-HER2 mAb (Carter, 1992) under control of the A0X1 promoter and containing the ARG1 selectable marker, named pGLY8135 ( Figure 13) .
  • This plasmid was transformed into the argl mutant GFI5.0 strains YGLY20599 (tupl-1) and YGLY4829 (TUP1) , and transformants were selected on medium lacking arginine . Positive clones were cultured in shake flasks for mAb production in a process similar to the 96 well plate procedure described
  • strains were inoculated into 50 ml of BMGY (Invitrogen, Carlsbad, CA) and cultured for 72h at 24 °C in a standard shaking incubator at 180 RPM, then the cells harvested by centrifugation and resuspended in 15 ml of BMMY
  • the YGLY20599 strain (tupl-1) mA -expressing clones yielded highly uniform complex human N-glycans (a mixture of afucosylated GO, Gl, and G2)
  • the YGLY4829 strain (TUP1) mAb-expressing clones yielded significantly more intermediate glycoforms, i.e. incompletely matured N-glycans containing hybrid and high mannose structures ( Figure 14) .
  • Example 9 Reversion of the wild type TUP1 gene in a
  • the P. pastoris mutant tupl-1 (1-137) gene including 500bp of the promoter region and 200bp of the terminator region, was PGR amplified from strain YGLY8316 genomic DNA using primers RCD916 5'- GCGGCCGCCCACAATGCTACTACAACACTCTTCCTG-3 1 and RCD917 5'-
  • Plasmid pGLY8l49 was digested with Hpal to linearize, and transformed into strain YGLY19193, which contains only the TUP1 wild type gene, and is a ura5 auxotroph (see Figure 8 and 17) .
  • Clones were selected on medium lacking uracil and screened by colony PCR and sequencing with primer RCD911 as shown in Example 4 to confirm integration of the plasmid.
  • One positive clone One positive clone,
  • YGLY26058 to complement the URA5 auxotrophy. Clones were selected on medium lacking uracil and two strains, confirmed to still contain only the tupl-1 mutant allele by PCR/sequencing (as in example 3) were saved and named YGLY26468 and YGLY26469.
  • Example 10 Reversion of the wild type TUPl gene back to the mutant tupl-1 allele restores increased antibody productivity and uniform N-glycans
  • Strain YGLY13992 (tupl-l) was cultured in micro24 5ml mini- fermenters (as described in Example 8) , along with TUPl-modified strains YGLY19250 (TUP1) , YGLY19251 (TUPl) , YGLY23502 (TUPl/tupl - 1) , YGLY26468 (tupl-l), and YGLY26469 (tupl-l) .
  • the cell -free culture supernatant was harvested by centrifugation at 2500 x g in a Beckman swinging bucket centrifuge and subjected to protein A purification by standard methods (Jiang, 2011) .
  • Antibody was quantified by analyzing on a Labchip GXII instrument ⁇ Caliper Life Sciences, Hopkinton, MA) using the standard HT Protein Express 200 method and calculated on a per liter basis.
  • the YGLY13992 tupl- 1 parental control strain produced 727 mg/L of secreted mAb, while the TUPl wild type complemented and TUPl/tupl- 1 heterozygous strains again produced significantly lower mAb titers, 60 +/- 13 mg/L and 184 +/- 61 mg/L, respectively ( Figure 18) .
  • the tupl-l reconstituted strain produced 530 mg/L, demonstrating restored mAb productivity in this tupl mutant strain and further establishing the direct impact of mutating TUPl on yeast secreted mAb productivity (Figure 18) . More over, after PNGaseF digestion, N-glycans released from mAb produced by strain YGLY26469 were again predominantly the human complex type (Figure 19) .
  • Example 11 Increased yield of secreted antibody through ectopic expression of a tupl-l mutant allele
  • Overexpression plasmids were constructed, first by PCR amplification for wild type TUPl full length (FL) open reading frame using primers RCD955 5'-
  • TCAATGAGCGTTCAAATTGGGAGGTGGC- 3 ' The TUPl FL and tupl-137 amplicons were cloned using the Topo 2.1 TA cloning vector (Invitrogen, Carlsbad, CA) , sequence verified, and subcloned into plasmid pGLY6301 (see Figure 20) , using the EcoRI/Fsel restriction sites, generating plasmid pGLY9894 ( Figure 21) and pGLY9895 ⁇ Figure 22) , respectively.
  • These plasmids contain the PpURA6 gene as a localization sequence for integration, the ScARR3 arsenite permease as a selectable marker (Wysocki, 1997) , and the AOX1 promoter driving the respective TUPl sequences.
  • Plasmids pGLY9894 and pGLY9895 were digested with Spel to linearize and transformed by electroporation into strain YGLY19250 and YGLY19251, and transformants selected on YSD medium (Standard recipe YPD with Soytone, Kerry Bio-Science, Rochester, N,
  • Strains YGLY19250 and YGLY19251 are GFI5.0
  • glycoengineered anti-HER2 mAb-producing strains that have been complemented to the wild type TUPl allele (See Figure 8 and Example 6) .
  • Positive clones were identified by PCR using primers AOXl-seq (5 ' - GCTTACTTTCATAATTGCGACTGGTTCC-3 ' ) and RCD966 (5 1 - GGCCGGCCTCAATGAGCGTTCAAATTGGGAGGTGGC-3 ' ) and then cultivated in micro24 5ml mini-fermenters (as described in Example 7) , along with parental TUPl wild type complemented strain YGLY19250 (TUPl) and parental control strain YGLY13992 (tupl-1) .
  • the cell-free culture supernatant was harvested by centrifugation at 2500 x g in a Beckman swinging bucket centrifuge and subjected to protein A purification by standard methods (Jiang, 2011) .
  • Antibody was quantified by
  • the YGLY13992 tupl-1 parental control strain produced 607 mg/L of secreted mAb, while the TUPl wild type complemented strain YGLY19250 produced significantly lower mAb titers, 130 +/- 8 mg/L ( Figure 23) .
  • the YGLY19250 clones overexpressing the AOXl-driven TUPl wild type gene produced even further reduced levels of mAb (75.6 +/- 28 mg/L), whereas the YGLY19250 clones overexpressing the AOXl-driven tupl-137 truncated allele produced increased levels of mAb (209.9 +/- 62 mg/L), though not to parental tupl-1 or reconstituted tupl-1 levels, indicating that the tupl-137 allele is partially dominant (Figure 23) .
  • plasmids pGLY9894 and pGLY9895 were digested with Spel to linearize and transformed by electroporation into strain YGLY4140, and transformants selected on YSD medium containing 0.3, 1, and 3 mM sodium arsenite.
  • Strain YGLY4140 is a glycoengineered strain that secretes proteins with the human Man 5 GlcNAc 2 N-glycan intermediate structure, and produces the secreted anti-HER2 mAb, and contains the wild type TUPl allele (Potgieter, 2008) .
  • the YGLY4140 TUPl wild type parental control strain produced 291 +/- 16 mg/L of secreted mAb, while the TUPl FL overexpressing clones produced 260 +/- 48 mg/L ⁇ Figure 24) .
  • the tupl- 137 overexpressing clones produced 512 +/- 116 mg/L, demonstrating that a tupl-l mutant allele acts in a dominant gain-of-function manner to impact mAb productivity in recombinant protein-producing yeast strains ( Figure 24) .
  • Example 1 The impact of TUPl mutation on recombinant protein-producing yeast is scalable
  • the cell-free culture supernatant was harvested by centrifugation at 2500 x g in a Beckman swinging bucket centrifuge and subjected to protein A purification by standard methods ⁇ Jiang, 2011) .
  • Antibody was quantified by reverse phase HPLC and calculated on a per liter basis.
  • the YGLY4140 TUPl wild type parental control strain produced 458 +/- 18 mg/L of secreted mAb, while the TUPl FL overexpressing clones produced only 230 +/- 26 mg/L, demonstrating that additional wild type TUPl actually has a deleterious affect on productivity in extended induction at larger scale (Figure 25) .
  • Example 13 The impact of TUPl mutation on recombinant protein-producing yeast is generally applicable to other
  • the plasmids TUPl overexpression plasmids pGLY9894 and pGLY9895 were digested with transformed into strain YGLY25818 as described above, and transformants selected on YSD medium containing 0.3, 1, and 3 m sodium arsenite.
  • Strain YGLY25818 is a glycoengineered strain that secretes proteins with the human intermediate Man 5 GlcNAc 2 glycoform (GFI2.0, Figure 7) and contains the plasmid pGLY4362 ( Figure 26) , which expresses a modified form of human pro-insulin containing a single N-glycosylation site in the B chain (B28N) of the protein as described in eehl (MRL-DOB-00062-US-PSP
  • Protein is secreted as a glycosylated single unprocessed polypeptide that runs at approximately 12kD on a reducing SDS-PAGE gel ( Figure 27, lane 1) .
  • Strain YGLY25818 along with one clone from YGLY25818 expressing the AOX1-TUP1 full length gene (YGLY26470) and two clones of YGLY25818 expressing the AOXl-tupl-137 truncated allele (YGLY26472, YGLY26473) were cultured in 1L Fedbatch Pro fermenters (DASGIP Biotools, Shrewsbury, MA) using a glycerol fedbatch followed by limiting-methanol feed induction process as previously described (Hopkins, 2011) .
  • Strains YGLY5819, YGLY5820, YGLY5821, YGLY5822, YGLY5827, and YGLY5828 are all prototrophic GFI5.0 strains that were constructed by transforming their respective parental argl auxotrophic GFI5.0 strains with an ARG1 marked plasmid that carries an AQXl-driven cassette containing a secreted MNSl derived from Trichoderma. reesei (see strain construction diagram in Figure 2) .
  • Strains YGLY5819 and YGLY5820, which derived from strain YGLY4828, are wild-type for TUP1.
  • strains YGLY5821 and YGLY5822 were derived from strain YGLY4829 and also contain the wild-type TUP1 allele.
  • strains YGLY5827 and YGLY5828 derived from strain YGLY4754 and as such contain the tupl-1 mutated allele.
  • strain YGLY13992 is a GFI5.0 strain with the tupl-1 allele, which was complemented to derive strain YGLY19193, which after re-introduction of URA5, yielded strains YGLY19250 and YGLY19251. Re- introduction of the tupl-1 mutant allele into strain YGLY19193 yielded strain YGLY26468. Strains YGLY13992, YGLY19250, YGLY19251, and YGLY26468 were struck for singles on YSD and YSD containing 0.2M CaCl 2 . Plates were incubated for 5 days at 24 °C then photographed.
  • Carter P Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL,

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Abstract

La présente invention concerne des cellules hôtes de levure exprimant un allèle muté de Tup1 ainsi que des procédés d'expression de polypeptides tels que des anticorps thérapeutiques, avec de telles cellules. Les souches exprimant l'allèle mutant de Tup1 ont présenté des niveaux d'expression supérieurs et une uniformité de glycane supérieure.
PCT/US2012/062231 2011-11-01 2012-10-26 Mutation de tup1 dans de la levure glycomodifiée WO2013066765A1 (fr)

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