公开(公告)号：US09845464B2

公开(公告)日：2017-12-19

申请号：US14517223

申请日：2014-10-17

申请人： GLYCOFI, INC.

发明人： Dongxing Zha ,  Stefan Wildt

IPC分类号： C40B40/08 ,  C12N15/10 ,  C07K16/00 ,  C07K16/28 ,  C07K16/32 ,  C07K16/40 ,  C40B40/02 ,  C40B50/06

CPC分类号： C12N15/1037 ,  C07K16/005 ,  C07K16/2887 ,  C07K16/32 ,  C07K16/40 ,  C07K2317/51 ,  C07K2317/55 ,  C12N15/1055 ,  C40B40/02 ,  C40B40/08 ,  C40B50/06

摘要： Methods for display of recombinant proteins or protein libraries on the surface of lower eukaryotes such as yeast and filamentous fungi are described. The methods are useful for screening libraries of recombinant proteins in lower eukaryotes to identify particular proteins with desired properties from the array of proteins in the libraries. The methods are particularly useful for constructing and screening antibody libraries in lower eukaryotes.

公开(公告)号：US10059952B2

公开(公告)日：2018-08-28

申请号：US14836203

申请日：2015-08-26

申请人： GLYCOFI, INC.

发明人： Juergen Nett

IPC分类号： C12N15/81 ,  C12N9/88

CPC分类号： C12N15/815 ,  C12N9/88 ,  C12N2800/102 ,  C12Y401/01021

摘要： Method and system for expression systems, based on ade1 and ade2 auxotrophic strains of yeast and fungi, including P. pastoris are disclosed. The expression systems are useful for increased cellular productivity of transformed cell lines and for production of recombinant glycoproteins at industrial scale.

公开(公告)号：US20150152427A1

公开(公告)日：2015-06-04

申请号：US14510249

申请日：2014-10-09

申请人： GLYCOFI, INC.

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

IPC分类号： C12N15/81 ,  C12N9/10 ,  C07K14/705 ,  C12N9/24

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

摘要： 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.

摘要翻译： 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：US20160068880A1

公开(公告)日：2016-03-10

申请号：US14927519

申请日：2015-10-30

申请人： GlycoFi, Inc.

发明人： Tillman U. Gerngross

IPC分类号： C12P21/00 ,  C12N9/24 ,  C12N15/81

CPC分类号： C12P21/005 ,  C07K2319/04 ,  C07K2319/05 ,  C12N1/14 ,  C12N9/1048 ,  C12N9/2488 ,  C12N15/79 ,  C12N15/80 ,  C12N15/81 ,  C12Y302/01 ,  C12Y302/01113

摘要： Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins I humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans such as Man5GlcNAc2 or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

摘要翻译： 已经开发了具有遗传修饰的糖基化途径的细胞系，其允许它们进行模拟I型糖蛋白的加工的酶反应序列。 在这些工程化宿主中表达的重组蛋白可以产生与其对应物更相似的糖蛋白（如果基本上不相同）。 低等真核生物通常产生含有甘露聚糖的高聚甘露，如Man5GlcNAc2或其他结构沿着人类糖基化途径。 这是使用以下工程和/或选择的组合实现的：不表达产生真菌糖蛋白特征的不合需要的复合结构的某些酶，其表达选择在真菌中存在的条件下具有最佳活性的外源酶 其中需要活性或靶向实现最佳活性的细胞器，以及其组合，其中遗传工程真核生物表达产生“人样”糖蛋白所需的多种外源酶。

公开(公告)号：US20150203890A1

公开(公告)日：2015-07-23

申请号：US14628392

申请日：2015-02-23

申请人： GlycoFi, Inc.

发明人： Stephen R. Hamilton

IPC分类号： C12P21/00

CPC分类号： C12P21/005 ,  C07K2319/036 ,  C07K2319/05 ,  C07K2319/055 ,  C12N9/1081 ,  C12N9/2402 ,  C12Y302/01018

摘要： The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and/or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. 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 sialylation) 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.

摘要翻译： 本发明涉及通过异源表达一组糖基转移酶（包括唾液酸转移酶和/或反式唾液酸酶）而被修饰以产生唾液酸化糖蛋白的真核宿主细胞，以成为用于产生哺乳动物例如人类治疗的宿主菌株 糖蛋白。 还提供了表达用于产生唾液酸化糖蛋白的CMP-唾液酸生物合成途径的新型真核宿主细胞。 本发明提供了可用于成功靶向并表达哺乳动物酶活性（例如参与唾液酸化的那些）的核酸分子和组合文库给真核宿主细胞中的细胞内区室。 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。

公开(公告)号：US20140051172A1

公开(公告)日：2014-02-20

申请号：US13956747

申请日：2013-08-01

申请人： GlycoFi, Inc.

发明人： Juergen H. Nett

IPC分类号： C12N15/81

CPC分类号： C12N15/905 ,  C07H21/04 ,  C07K2319/00 ,  C12N9/1077 ,  C12N15/81 ,  C12N15/815

摘要： A novel gene encoding P. pastoris orotate-phosphoribosyl transferase (URA5) is disclosed. Methods for producing and selecting yeast strains capable of stable genetic integration of heterologous sequences into the host genome are also provided.

摘要翻译： 公开了编码巴斯德毕赤酵母 - 磷酸核糖转移酶（URA5）的新型基因。 还提供了用于产生和选择能够将异源序列稳定遗传整合到宿主基因组中的酵母菌株的方法。

公开(公告)号：US20150087013A1

公开(公告)日：2015-03-26

申请号：US14567459

申请日：2014-12-11

申请人： GLYCOFI, INC.

发明人： Juergen H. Nett

IPC分类号： C12N15/90

CPC分类号： C12N15/905 ,  C07H21/04 ,  C07K2319/00 ,  C12N9/1077 ,  C12N15/81 ,  C12N15/815

摘要： A novel gene encoding P. pastoris orotate-phosphoribosyl transferase (URA5) is disclosed. Methods for producing and selecting yeast strains capable of stable genetic integration of heterologous sequences into the host genome are also provided.

摘要翻译： 公开了编码巴斯德毕赤酵母 - 磷酸核糖转移酶（URA5）的新型基因。 还提供了用于产生和选择能够将异源序列稳定遗传整合到宿主基因组中的酵母菌株的方法。

公开(公告)号：US20150051381A1

公开(公告)日：2015-02-19

申请号：US14454044

申请日：2014-08-07

申请人： GlycoFi, Inc.

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

IPC分类号： C12P21/00 ,  C07K14/47

CPC分类号： C12P21/005 ,  C07K14/47 ,  C12N9/1051 ,  C12N15/1082 ,  C12N15/79

摘要： 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.

摘要翻译： 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：US08771989B2

公开(公告)日：2014-07-08

申请号：US13938603

申请日：2013-07-10

申请人： GlycoFi, Inc.

发明人： Byung-Kwon Choi

IPC分类号： C07K16/00 ,  C12P21/00 ,  C12N1/15 ,  C12N1/19 ,  C12N15/81

CPC分类号： C07K16/00 ,  C12N15/815 ,  C12P21/005

摘要： Lower eukaryote host cells in which an endogenous or heterologous Ca2+ ATPase is overexpressed are described. Also described are lower eukaryote host cells in which a calreticulin and/or ERp57 protein are overexpressed. These host cells are useful for producing recombinant glycoproteins that have reduced O-glycosylation.

摘要翻译： 描述了内源或异源Ca2 + ATP酶过表达的低等真核生物宿主细胞。 还描述了其中钙网蛋白和/或ERp57蛋白过表达的低等真核细胞宿主细胞。 这些宿主细胞可用于产生具有降低的O-糖基化的重组糖蛋白。

公开(公告)号：US20130316403A1

公开(公告)日：2013-11-28

申请号：US13938603

申请日：2013-07-10

申请人： GlycoFi, Inc.

发明人： Byung-Kwon Choi

IPC分类号： C07K16/00

CPC分类号： C07K16/00 ,  C12N15/815 ,  C12P21/005

摘要： Lower eukaryote host cells in which an endogenous or heterologous Ca2+ ATPase is overexpressed are described. Also described are lower eukaryote host cells in which a calreticulin and/or ERp57 protein are overexpressed. These host cells are useful for producing recombinant glycoproteins that have reduced O-glycosylation.

摘要翻译： 描述了内源或异源Ca2 + ATP酶过表达的低等真核生物宿主细胞。 还描述了其中钙网蛋白和/或ERp57蛋白过表达的低等真核细胞宿主细胞。 这些宿主细胞可用于产生具有降低的O-糖基化的重组糖蛋白。