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    PRODUCTION OF MODIFIED GLYCOPROTEINS HAVING MULTIPLE ANTENNARY STRUCTURES
    2.
    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。

    METHODS FOR REDUCING OR ELIMINATING ALPHA-MANNOSIDASE RESISTANT GLYCANS IN THE PRODUCTION OF GLYCOPROTEINS
    4.
    发明申请
    METHODS FOR REDUCING OR ELIMINATING ALPHA-MANNOSIDASE RESISTANT GLYCANS IN THE PRODUCTION OF GLYCOPROTEINS 审中-公开
    用于减少或消除糖尿病抗性糖蛋白在糖蛋白生产中的方法

    公开(公告)号：WO2005106010A2

    公开(公告)日：2005-11-10

    申请号：PCT/IB2005/051415

    申请日：2005-04-29

    Applicant: GLYCOFI, INC. ,  BOBROWICZ, Piotr

    Inventor： BOBROWICZ, Piotr

    IPC: C12P21/06

    CPC classification number: C07K14/39 ,  C12N9/1051 ,  C12N15/80 ,  C12P21/005

    Abstract: The present invention provides methods to reduce or eliminate a-mannosidase resistant glycans on glycoproteins in yeast. The reduction or elimination of a-mannosidase resistant glycans on glycoproteins results from the disruption of the newly isolated P. pastoris AMR 2 gene encoding β1,2-mannosyltransferase. The present invention also discloses novel genes, polypeptides, antibodies, vectors and host cells relating to a-mannosidase resistance on glycans.

    Abstract translation: 本发明提供在酵母中减少或消除糖蛋白上的α-甘露糖苷酶抗性聚糖的方法。 糖蛋白上的α-甘露糖苷酶抗性聚糖的还原或消除是由新分离的编码β1,2-甘露糖基转移酶的巴斯德毕赤酵母AMR2基因的破坏引起的。 本发明还公开了与聚糖上的α-甘露糖苷酶抗性有关的新基因，多肽，抗体，载体和宿主细胞。

    COMBINATORIAL DNA LIBRARY FOR PRODUCING MODIFIED N-GLYCANS IN LOWER EUKARYOTES
    5.
    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。

    METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS
    7.
    发明申请
    METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS 审中-公开
    葡萄糖生物化学烟草中糖基化途径的代谢工程

    公开(公告)号：WO2010099153A3

    公开(公告)日：2010-10-21

    申请号：PCT/US2010025163

    申请日：2010-02-24

    Applicant: MERCK SHARP & DOHME ,  DAVIDSON ROBERT C ,  BOBROWICZ PIOTR ,  ZHA DONGXING

    Inventor： DAVIDSON ROBERT C ,  BOBROWICZ PIOTR ,  ZHA DONGXING

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

    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已经根据本文的方法进行遗传工程化的低等真核细胞，例如巴斯德毕赤酵母，其使用半乳糖作为唯一的碳源。 细胞被遗传工程化以表达几种包含Leloir途径的酶。 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-磷酸尿苷酸转移酶，以及任选的半乳糖通透酶。 此外，提供了一种用于提高具有半乳糖封端或含有N-聚糖的糖蛋白的产率的方法，所述糖蛋白在已被遗传工程化以产生具有半乳糖残基的N-聚糖的糖蛋白的细胞中，但通常缺少包含Leloir的酶 途径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-磷酸尿苷转移酶的一种或多种核酸分子转化细胞。 描述的方法和宿主细胞使得能够存在或缺乏同化半乳糖的能力作为制备重组细胞的选择方法。 本文显示的方法和宿主细胞特别可用于制备具有半乳糖终止或含有N-聚糖的免疫球蛋白等。

    N-ACETYLGLUCOSAMINYLTRANSFERASE III EXPRESSION IN LOWER EUKARYOTES
    9.
    发明申请
    N-ACETYLGLUCOSAMINYLTRANSFERASE III EXPRESSION IN LOWER EUKARYOTES 审中-公开
    较低级别的N-乙酰胆碱氨基转移酶III表达

    公开(公告)号：WO2004074458A2

    公开(公告)日：2004-09-02

    申请号：PCT/US2004/005128

    申请日：2004-02-20

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

    Inventor： BOBROWICZ, Piotr ,  HAMILTON, Stephen, R. ,  GERNGROSS, Tillman, 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 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 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 activity, which produce bisected N -glycan structures and may 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。 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。 产生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活性，其产生二等分N-聚糖结构，并且可以进一步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，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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