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    RECOMBINANT ECTODOMAIN EXPRESSION OF HERPES SIMPLEX VIRUS GLYCOPROTEINS IN YEAST
    2.
    发明申请
    RECOMBINANT ECTODOMAIN EXPRESSION OF HERPES SIMPLEX VIRUS GLYCOPROTEINS IN YEAST 审中-公开
    HERPES的重组ECTODOMAIN表达在YEAST中的简单病毒糖蛋白

    公开(公告)号：WO2011011495A1

    公开(公告)日：2011-01-27

    申请号：PCT/US2010/042719

    申请日：2010-07-21

    Applicant: MERCK SHARP & DOHME CORP. ,  BRYAN, Janine, T. ,  BALLIET, John, W. ,  FLYNN, Jessica, A. ,  CASIMIRO, Danilo, R. ,  DAVIDSON, Robert, C. ,  COPELAND, Victoria ,  RIOS, Sandra, E. ,  CHOI, Byun-Kwon ,  WILDT, Stefan

    Inventor： BRYAN, Janine, T. ,  BALLIET, John, W. ,  FLYNN, Jessica, A. ,  CASIMIRO, Danilo, R. ,  DAVIDSON, Robert, C. ,  COPELAND, Victoria ,  RIOS, Sandra, E. ,  CHOI, Byun-Kwon ,  WILDT, Stefan

    IPC: A61K39/245 ,  C07K1/00

    CPC classification number: A61K39/245 ,  A61K39/12 ,  A61K2039/55505 ,  A61K2039/55577 ,  A61K2039/57 ,  C12N2710/16622 ,  C12N2710/16634

    Abstract: The present invention provides Herpes Simplex Virus (HSV) gD, gC, gB and/or gE recombinant glycoproteins having a particular pre-selected N-linked glycosylation pattern as the predominant N-glycoform. The present invention also provides methods of producing these recombinant glycoproteins in yeast, preferably Pichia pastoris , which may be glycoengineered to provide particular glycosylation patterns. The present invention further provides vaccines comprising gD and gC, and optionally gB and/or gE, at least one of which has a particular pre-selected N-linked glycosylation pattern as the predominant N-glycoform. The recombinant glycoproteins are produced by a method which, in one embodiment, comprises transforming a yeast of the genus Pichia with an expression vector containing a DNA encoding an HSV glycoprotein, which is under regulation of a promoter functional in a yeast of the genus Pichia, culturing the yeast in a medium, and recovering the recombinant glycoprotein from the obtained culture. DNA encoding the recombinant glycoproteins is preferably codon-optimized to achieve optimal expression in Pichia .

    Abstract translation: 本发明提供具有特定预先选择的N-连接的糖基化模式作为主要N-糖基的单纯疱疹病毒（HSV）gD，gC，gB和/或gE重组糖蛋白。 本发明还提供了在酵母，优选巴斯德毕赤酵母中生产这些重组糖蛋白的方法，其可以糖工程化以提供特定的糖基化模式。 本发明还提供了包含gD和gC以及任选的gB和/或gE的疫苗，其中至少一种具有特定的预先选择的N-连接的糖基化模式作为主要的N-糖基型。 通过一种方法制备重组糖蛋白，该方法在一个实施方案中包括用含有编码HSV糖蛋白的DNA的表达载体转化毕赤酵母属的酵母，所述HSV糖蛋白处于毕赤酵母属酵母中起促进作用的启动子的调节下， 在培养基中培养酵母，并从获得的培养物中回收重组糖蛋白。 编码重组糖蛋白的DNA优选密码子优化以在毕赤酵母中达到最佳表达。

    METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS
    3.
    发明申请
    METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS 审中-公开
    糖生成酵母菌毕赤酵母中半乳糖同工酶途径的代谢工程

    公开(公告)号：WO2010099153A2

    公开(公告)日：2010-09-02

    申请号：PCT/US2010/025163

    申请日：2010-02-24

    Applicant: MERCK SHARP & DOHME CORP. ,  DAVIDSON, Robert, C. ,  BOBROWICZ, Piotr ,  ZHA, Dongxing

    Inventor： DAVIDSON, Robert, C. ,  BOBROWICZ, Piotr ,  ZHA, Dongxing

    IPC: C12N15/81 ,  C12N15/54 ,  C12N15/61 ,  C12N9/12 ,  C12N9/90 ,  C12N1/16 ,  C12P21/00 ,  C07K16/18

    CPC classification number: C12P21/005 ,  C07K16/32 ,  C07K2317/14 ,  C07K2317/41 ,  C12N1/16 ,  C12N9/1205 ,  C12N9/1241 ,  C12N9/90 ,  C12N15/815

    Abstract: Lower eukaryotic cells such as Pichia pastoris that normally cannot use galactose as a carbon source but which have been genetically engineered according to the methods herein to use galactose as a sole source of carbon are described. The cells are genetically engineered to express several of the enzymes comprising the Leloir pathway. In particular, the cells are genetically engineered to express a galactokinase, a UDP-galactose-C4-epimerase, and a galactose-1-phosphate uridyltransferase, and optionally a galactose permease. In addition, a method is provided for improving the yield of glycoproteins that have galactose-terminated or -containing N-glycans in cells that have been genetically engineered to produce glycoproteins with N-glycans having galactose residues but which normally lack the enzymes comprising the Leloir pathway comprising transforming the cells with one or more nucleic acid molecules encoding a galactokinase, a UDP-galactose-C4-epimerase, and a galactose-1-phosphate uridyltransferase. The methods and host cells described enable the presence or lack of the ability to assimilate galactose as a selection method for making recombinant cells. The methods and host cells are shown herein to be particularly useful for making immunoglobulins and the like that have galactose-terminated or containing N-glycans.

    Abstract translation: 描述了通常不能使用半乳糖作为碳源但是已经根据本文的方法基因工程化以使用半乳糖作为碳的唯一来源的低等真核细胞，例如巴斯德毕赤酵母（Pichia pastoris）。 这些细胞经过遗传工程改造后可以表达包含Leloir途径的几种酶。 特别地，细胞被遗传工程化以表达半乳糖激酶，UDP-半乳糖-C4-差向异构酶和半乳糖-1-磷酸尿苷酰转移酶，并且任选地表达半乳糖通透酶。 此外，提供了一种方法，用于提高细胞中具有半乳糖封端或含有N-聚糖的糖蛋白的产量，所述细胞经基因工程改造以产生具有半乳糖残基的N-聚糖但通常缺少包含Leloir的酶的糖蛋白 包括用编码半乳糖激酶，UDP-半乳糖-C4-差向异构酶和半乳糖-1-磷酸尿苷酰转移酶的一种或多种核酸分子转化细胞。 所描述的方法和宿主细胞使得存在或缺乏同化半乳糖作为制备重组细胞的选择方法的能力。 这里显示的方法和宿主细胞对于制备具有半乳糖封端的或含有N-聚糖的免疫球蛋白等是特别有用的。

    PRODUCTION OF MODIFIED GLYCOPROTEINS HAVING MULTIPLE ANTENNARY STRUCTURES
    5.
    发明申请
    PRODUCTION OF MODIFIED GLYCOPROTEINS HAVING MULTIPLE ANTENNARY STRUCTURES 审中-公开
    生产具有多种天然结构的改性糖蛋白

    公开(公告)号：WO2004074461A2

    公开(公告)日：2004-09-02

    申请号：PCT/US2004/005191

    申请日：2004-02-20

    Applicant: BOBROWICZ, Piotr ,  HAMILTON, Stephen, R. ,  GERNGROSS, Tilman, U. ,  WILDT, Stefan ,  CHOI, Byung-Kwon ,  NETT, Juergen, Hermann ,  DAVIDSON, Robert, C.

    Inventor： BOBROWICZ, Piotr ,  HAMILTON, Stephen, R. ,  GERNGROSS, Tilman, U. ,  WILDT, Stefan ,  CHOI, Byung-Kwon ,  NETT, Juergen, Hermann ,  DAVIDSON, Robert, C.

    IPC: C12N

    CPC classification number: C07K14/39 ,  A61K38/00 ,  C07K2319/05 ,  C12N9/1051 ,  C12P21/005 ,  Y02A50/396

    Abstract: The present invention relates to eukaryotic host cells, especially lower eukaryotic host cells, having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar and sugar nucleotide transporters to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII, GnTIV, GnTV, GnT VI or GnTIX activity, which produce bisected and/or multiantennary N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar, sugar nucleotide transporters, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

    Abstract translation: 本发明涉及具有修饰的寡糖的真核宿主细胞，特别是较低的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖和糖核苷酸转运蛋白进一步修饰，以成为用于产生哺乳动物的宿主菌株， 人类治疗糖蛋白。 该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。 制备或选择具有修饰的脂质连接寡糖的宿主细胞。 在工程化宿主细胞中制备的N-聚糖表现出GnTIII，GnTIV，GnTV，GnT VI或GnTIX活性，其产生二等分和/或多元N-聚糖结构，并且可以通过异源表达一种或多种酶，例如糖基转移酶 ，糖，糖核苷酸转运蛋白，以产生人样糖蛋白。 为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

    PRODUCTION OF MODIFIED GLYCOPROTEINS HAVING MULTIPLE ANTENNARY STRUCTURES
    6.
    发明申请
    PRODUCTION OF MODIFIED GLYCOPROTEINS HAVING MULTIPLE ANTENNARY STRUCTURES 审中-公开
    生产具有多种天然结构的改性糖蛋白

    公开(公告)号：WO2004074461A3

    公开(公告)日：2005-03-17

    申请号：PCT/US2004005191

    申请日：2004-02-20

    Applicant: BOBROWICZ PIOTR ,  HAMILTON STEPHEN R ,  GERNGROSS TILMAN U ,  WILDT STEFAN ,  CHOI BYUNG-KWON ,  NETT JUERGEN HERMANN ,  DAVIDSON ROBERT C

    Inventor： BOBROWICZ PIOTR ,  HAMILTON STEPHEN R ,  GERNGROSS TILMAN U ,  WILDT STEFAN ,  CHOI BYUNG-KWON ,  NETT JUERGEN HERMANN ,  DAVIDSON ROBERT C

    IPC: A61K38/00 ,  C07K14/39 ,  C07K14/47 ,  C12N1/18 ,  C12N9/10 ,  C12N15/12 ,  C12N15/74 ,  C12P21/00 ,  C12P21/06 ,  C12N5/10 ,  C12N15/81

    CPC classification number: C07K14/39 ,  A61K38/00 ,  C07K2319/05 ,  C12N9/1051 ,  C12P21/005

    Abstract: The present invention relates to eukaryotic host cells, especially lower eukaryotic host cells, having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar and sugar nucleotide transporters to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII, GnTIV, GnTV, GnT VI or GnTIX activity, which produce bisected and/or multiantennary N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar, sugar nucleotide transporters, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

    Abstract translation: 本发明涉及具有修饰的寡糖的真核宿主细胞，特别是较低的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖和糖核苷酸转运蛋白进一步修饰，以成为用于产生哺乳动物的宿主菌株， 人类治疗糖蛋白。 该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。 制备或选择具有修饰的脂质连接寡糖的宿主细胞。 在工程化的宿主细胞中制备的N-聚糖表现出GnTIII，GnTIV，GnTV，GnT VI或GnTIX活性，其产生二等分和/或多元N-聚糖结构，并且可以通过异源表达一种或多种酶，例如糖基转移酶 ，糖，糖核苷酸转运蛋白，以产生人样糖蛋白。 为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

    COMBINATORIAL DNA LIBRARY FOR PRODUCING MODIFIED N-GLYCANS IN LOWER EUKARYOTES
    10.
    发明申请
    COMBINATORIAL DNA LIBRARY FOR PRODUCING MODIFIED N-GLYCANS IN LOWER EUKARYOTES 审中-公开
    用于生产修复的N-GLYCANS在下一代的组合DNA文库

    公开(公告)号：WO2004074499A3

    公开(公告)日：2005-01-27

    申请号：PCT/US2004005244

    申请日：2004-02-20

    Applicant: GERNGROSS TILLMAN U ,  WILDT STEFAN ,  CHOI BYUNG-KWON ,  NETT JUERGEN HERMANN ,  BOBROWICZ PIOTR ,  HAMILTON STEPHEN R ,  DAVIDSON ROBERT C

    Inventor： GERNGROSS TILLMAN U ,  WILDT STEFAN ,  CHOI BYUNG-KWON ,  NETT JUERGEN HERMANN ,  BOBROWICZ PIOTR ,  HAMILTON STEPHEN R ,  DAVIDSON ROBERT C

    IPC: C12N9/10 ,  C12N15/79 ,  C12P21/00 ,  C12N1/14 ,  C12N5/10 ,  C12N9/24 ,  C12R1/645

    CPC classification number: C12P21/005 ,  C07K14/47 ,  C12N9/1051 ,  C12N15/1082 ,  C12N15/79

    Abstract: The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities such as those involved in glycosylation to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man5GlcNAc2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

    Abstract translation: 本发明涉及具有修饰的寡糖的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖转运体和甘露糖苷酶进一步修饰，以成为用于产生哺乳动物例如人治疗性糖蛋白的宿主菌株。 本发明提供核酸分子和组合文库，其可用于成功靶向和表达哺乳动物酶活性，例如参与糖基化的真核宿主细胞中的细胞内区室的那些。 该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。 建立或选择具有修饰寡糖的宿主细胞。 在工程化宿主细胞中制备的N-聚糖具有Man5GlcNAc2核心结构，然后可以通过异源表达一种或多种酶，例如糖基转移酶，糖转运蛋白和甘露糖苷酶来进一步修饰，以产生人样糖蛋白。 为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

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