公开(公告)号：US20130217067A1

公开(公告)日：2013-08-22

申请号：US13408432

申请日：2012-02-29

Applicant: TILLMAN U. GERNGROSS ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

Inventor： TILLMAN U. GERNGROSS ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

IPC: C07K14/47

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.

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。

公开(公告)号：US20120052530A1

公开(公告)日：2012-03-01

申请号：US13156804

申请日：2011-06-09

Applicant: TILLMAN U. GERNGROSS ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

Inventor： TILLMAN U. GERNGROSS ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

IPC: C12P21/00 ,  C12N1/19 ,  C40B40/08

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。

公开(公告)号：US20100016555A1

公开(公告)日：2010-01-21

申请号：US12540915

申请日：2009-08-13

Applicant: Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen H. Nett ,  Robert C. Davidson

Inventor： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen H. Nett ,  Robert C. Davidson

IPC: C07K16/00 ,  C07K14/435

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。 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活性，其产生二等分的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，糖转运蛋白和甘露糖苷酶）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。

公开(公告)号：US20090226959A1

公开(公告)日：2009-09-10

申请号：US12313636

申请日：2008-11-21

Applicant: Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

Inventor： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

IPC: C12P21/00 ,  C12N1/19 ,  C12N1/15 ,  C12N15/63 ,  C07K2/00

CPC classification number: C12P21/005 ,  C12N9/1051

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。

公开(公告)号：US08932825B2

公开(公告)日：2015-01-13

申请号：US10500240

申请日：2002-12-24

Applicant: Stefan Wildt ,  Robert Gordon Miele ,  Juergen Hermann Nett ,  Robert C. Davidson

Inventor： Stefan Wildt ,  Robert Gordon Miele ,  Juergen Hermann Nett ,  Robert C. Davidson

IPC: G01N33/569 ,  C12P1/02 ,  C12N1/00 ,  A61K51/00 ,  C12N9/10 ,  C12P21/00

CPC classification number: C12N15/815 ,  A01K2217/075 ,  C07K14/705 ,  C07K2319/05 ,  C12N9/1051 ,  C12N9/2402 ,  C12P21/005 ,  C12Y204/01143 ,  C12Y204/01144 ,  C12Y204/01145 ,  C12Y204/01155 ,  C12Y302/01024

Abstract: The present invention relates to host cells having modified lipid-linked 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 have a GlcNAcMan3GlcNAc2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, 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: 本发明涉及具有经修饰的脂质连接的寡糖的宿主细胞，其可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。 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-聚糖具有GlcNAcMan3GlcNAc2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。

公开(公告)号：US08877462B2

公开(公告)日：2014-11-04

申请号：US13408432

申请日：2012-02-29

Applicant: Tillman U. Gerngross ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

Inventor： Tillman U. Gerngross ,  Stefan Wildt ,  Byung-kwon Choi ,  Juergen Hermann Nett ,  Piotr Bobrowicz ,  Stephen R. Hamilton ,  Robert C. Davidson

IPC: C12N1/14

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.

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。

公开(公告)号：US08697394B2

公开(公告)日：2014-04-15

申请号：US10546101

申请日：2004-02-20

Applicant: Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

Inventor： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

IPC: C12P21/02 ,  C12N1/00 ,  C12N1/15 ,  C12N15/00

CPC classification number: C12P21/005 ,  C12N9/1051

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。

公开(公告)号：US20120121630A1

公开(公告)日：2012-05-17

申请号：US13386508

申请日：2010-07-21

Applicant: Janine T. Bryan ,  John W. Ballet ,  Jessica A. Flynn ,  Danilo R. Casimiro ,  Robert C. Davidson ,  Victoria Copeland ,  Sandra E. Rios ,  Byung-Kwon Choi ,  Stefan Wildt

Inventor： Janine T. Bryan ,  John W. Ballet ,  Jessica A. Flynn ,  Danilo R. Casimiro ,  Robert C. Davidson ,  Victoria Copeland ,  Sandra E. Rios ,  Byung-Kwon Choi ,  Stefan Wildt

IPC: A61K39/245 ,  A61P37/06 ,  C12N15/38 ,  C07K14/035 ,  C12P21/00

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-糖型的单纯疱疹病毒（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，其可以进行糖工程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。 重组糖蛋白通过一种方法产生，在一个实施方案中，该方法包括用含有编码HSV糖蛋白的DNA的表达载体转化毕赤酵母属的酵母，所述HSV糖蛋白在毕赤酵母属酵母中起促进作用的启动子的调控， 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。

公开(公告)号：US20120003695A1

公开(公告)日：2012-01-05

申请号：US13202002

申请日：2010-02-24

Applicant: Robert C. Davidson ,  Piotr Bobrowicz ,  Dongxing Zha

Inventor： Robert C. Davidson ,  Piotr Bobrowicz ,  Dongxing Zha

IPC: C12P21/00 ,  C12N9/64 ,  C12N15/81 ,  C12N9/16 ,  C12N9/24 ,  C12N1/19 ,  C12N9/72

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。 所描述的方法和宿主细胞使得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。

公开(公告)号：US20110086054A1

公开(公告)日：2011-04-14

申请号：US12995284

申请日：2009-12-03

Applicant: Natarajan Sethuraman ,  Robert C. Davidson ,  Terrance A. Stadheim ,  Stefan Wildt

Inventor： Natarajan Sethuraman ,  Robert C. Davidson ,  Terrance A. Stadheim ,  Stefan Wildt

IPC: A61K39/00 ,  C12N1/19 ,  C12N1/15 ,  C12N15/80 ,  C12N15/81 ,  C07K14/00 ,  A61P37/04

CPC classification number: C07K14/435 ,  C12N15/80 ,  C12P21/005

Abstract: Lower eukaryotic host cells have been engineered to produce glycoprotein having at least one terminal α-galactosyl epitope. The glycoproteins are useful for the production of highly antigenic glycoprotein compositions with advantages for the production of vaccines.

Abstract translation: 已经将低等真核宿主细胞工程化以产生具有至少一个末端α-半乳糖基表位的糖蛋白。 糖蛋白可用于生产具有疫苗生产优点的高抗原性糖蛋白组合物。