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1. 发明申请

WO2013062940A1 ENGINEERED LOWER EUKARYOTIC HOST STRAINS FOR RECOMBINANT PROTEIN EXPRESSION 审中-公开
标题翻译：用于重组蛋白表达的工程化下一代真菌宿主菌株
公开(公告)号：WO2013062940A1
公开(公告)日：2013-05-02
申请号：PCT/US2012/061432
申请日：2012-10-23
申请人： MERCK SHARP & DOHME CORP. , JIANG, Bo , ARGYROS, Rebecca, D. , NELSON, Stephanie , DAVIDSON, Robert, C. , CHEN, Ronghua , ZHUANG, Jun
发明人： JIANG, Bo , ARGYROS, Rebecca, D. , NELSON, Stephanie , DAVIDSON, Robert, C. , CHEN, Ronghua , ZHUANG, Jun
IPC分类号： C12N15/09 , C07H21/04
CPC分类号： C12P21/00 , C07K14/39 , C07K16/32 , C07K2317/14 , C12N15/80 , C12P21/005
摘要： The present invention relates to novel engineered lower eukaryotic host cells for expressing heterologous proteins and to methods of generating such strains.
摘要翻译：本发明涉及用于表达异源蛋白质的新型工程化低级真核宿主细胞和产生这种菌株的方法。

2. 发明申请

WO2011011495A1 RECOMBINANT ECTODOMAIN EXPRESSION OF HERPES SIMPLEX VIRUS GLYCOPROTEINS IN YEAST 审中-公开
标题翻译： 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
申请人： 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
发明人： 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分类号： A61K39/245 , C07K1/00
CPC分类号： A61K39/245 , A61K39/12 , A61K2039/55505 , A61K2039/55577 , A61K2039/57 , C12N2710/16622 , C12N2710/16634
摘要： 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 .
摘要翻译：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。

3. 发明申请

WO2010099153A2 METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS 审中-公开
标题翻译：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
申请人： MERCK SHARP & DOHME CORP. , DAVIDSON, Robert, C. , BOBROWICZ, Piotr , ZHA, Dongxing
发明人： DAVIDSON, Robert, C. , BOBROWICZ, Piotr , ZHA, Dongxing
IPC分类号： C12N15/81 , C12N15/54 , C12N15/61 , C12N9/12 , C12N9/90 , C12N1/16 , C12P21/00 , C07K16/18
CPC分类号： C12P21/005 , C07K16/32 , C07K2317/14 , C07K2317/41 , C12N1/16 , C12N9/1205 , C12N9/1241 , C12N9/90 , C12N15/815
摘要： 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.
摘要翻译：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。

4. 发明公开

EP2456463A1 RECOMBINANT ECTODOMAIN EXPRESSION OF HERPES SIMPLEX VIRUS GLYCOPROTEINS IN YEAST 审中-公开
标题翻译：酵母中的单纯疱疹病毒糖蛋白的重组EKTODOMÄNENEXPRESSION
公开(公告)号：EP2456463A1
公开(公告)日：2012-05-30
申请号：EP10802836.6
申请日：2010-07-21
申请人： Merck Sharp & Dohme Corp.
发明人： 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分类号： A61K39/245 , C07K1/00
CPC分类号： A61K39/245 , A61K39/12 , A61K2039/55505 , A61K2039/55577 , A61K2039/57 , C12N2710/16622 , C12N2710/16634
摘要： 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 .

5. 发明公开

EP2401378A2 METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS 有权
标题翻译：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：EP2401378A2
公开(公告)日：2012-01-04
申请号：EP10706438.8
申请日：2010-02-24
申请人： Merck Sharp & Dohme Corp.
发明人： DAVIDSON, Robert, C. , BOBROWICZ, Piotr , ZHA, Dongxing
IPC分类号： C12N15/81 , C12N15/54 , C12N15/61 , C12N9/12 , C12N9/90 , C12N1/16 , C12P21/00 , C07K16/18
CPC分类号： C12P21/005 , C07K16/32 , C07K2317/14 , C07K2317/41 , C12N1/16 , C12N9/1205 , C12N9/1241 , C12N9/90 , C12N15/815
摘要： 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.
摘要翻译：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改造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。

6. 发明授权

EP2401378B1 METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS 有权
标题翻译：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：EP2401378B1
公开(公告)日：2013-08-14
申请号：EP10706438.8
申请日：2010-02-24
申请人： Merck Sharp & Dohme Corp.
发明人： DAVIDSON, Robert, C. , BOBROWICZ, Piotr , ZHA, Dongxing
IPC分类号： C12N15/81 , C12N15/54 , C12N15/61 , C12N9/12 , C12N9/90 , C12N1/16 , C12P21/00 , C07K16/18
CPC分类号： C12P21/005 , C07K16/32 , C07K2317/14 , C07K2317/41 , C12N1/16 , C12N9/1205 , C12N9/1241 , C12N9/90 , C12N15/815

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