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1. 发明授权

US08372636B2 Yeast cell surface display of proteins and uses thereof 有权
公开(公告)号：US08372636B2
公开(公告)日：2013-02-12
申请号：US12316916
申请日：2008-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12N15/00
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

2. 发明授权

US06696251B1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06696251B1
公开(公告)日：2004-02-24
申请号：US09724108
申请日：2000-11-28
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12Q168
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可显示约10 4个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，而没有来自细胞壁组分的空间位阻。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

3. 发明申请

US20090280560A1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US20090280560A1
公开(公告)日：2009-11-12
申请号：US12316916
申请日：2008-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12N15/63 , C07K9/00 , C07K16/00 , C07K14/725
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可以显示大约104个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，而没有来自细胞壁组分的空间位阻。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

4. 发明授权

US07465787B2 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US07465787B2
公开(公告)日：2008-12-16
申请号：US10738454
申请日：2003-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C07K14/37 , C07K14/39 , C07K16/28 , C07K16/46 , C07K17/00 , C07K17/02 , C07K17/06 , C12Q1/02 , C12Q1/68 , C12N1/19 , C12N15/63 , C12N15/64
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 10 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可以显示约10个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，而没有来自细胞壁组件的空间位阻。

5. 发明授权

US06699658B1 Yeast cell surface display of proteins and uses thereof 失效
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06699658B1
公开(公告)日：2004-03-02
申请号：US09009388
申请日：1998-01-20
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12Q168
CPC分类号： C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance, from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可显示约10 4个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，没有空间位阻，来自细胞壁组分。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

6. 发明授权

US06331391B1 Yeast cell surface display of proteins and uses thereof 失效
公开(公告)号：US06331391B1
公开(公告)日：2001-12-18
申请号：US09009388
申请日：1998-01-20
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12Q168
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

7. 发明授权

US06423538B1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06423538B1
公开(公告)日：2002-07-23
申请号：US09724297
申请日：2000-11-28
申请人： K. Dane Wittrup , David M. Kranz , Michele Keike , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Keike , Eric T. Boder
IPC分类号： C12N1581
CPC分类号： C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. If effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可以显示大约104个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。如果发挥作用，酵母已经发展出蛋白质 - 蛋白质结合的平台，没有细胞壁组分的空间位阻。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

8. 发明授权

US06300065B1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06300065B1
公开(公告)日：2001-10-09
申请号：US09140084
申请日：1998-08-26
申请人： Michele C. Kieke , K. Dane Wittrup , Eric T. Boder , David M. Kranz , Eric Shusta
发明人： Michele C. Kieke , K. Dane Wittrup , Eric T. Boder , David M. Kranz , Eric Shusta
IPC分类号： C12P2102
CPC分类号： C40B40/02 , C12N15/1037 , G01N33/68 , G01N33/6845 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. Attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening. As another embodiment, the selection method identifies proteins displayed at higher levels as proteins that are secreted at higher efficiency and that are more stable.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内亲和力成熟。 T细胞受体突变体可通过这种有效显示在酵母细胞表面上的方法分离，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。作为另一个实施方案，选择方法鉴定在较高水平上显示的蛋白质作为更高效率分泌并且更稳定的蛋白质。

9. 发明授权

US07569357B2 High affinity TCR proteins and methods 有权
标题翻译：高亲和力TCR蛋白和方法
公开(公告)号：US07569357B2
公开(公告)日：2009-08-04
申请号：US10783786
申请日：2004-02-20
申请人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
发明人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
IPC分类号： G01N33/563 , G01N33/53
CPC分类号： C40B40/02 , C07K14/7051 , C12N15/1037 , G01N33/68 , G01N33/6842 , G01N33/6854 , G01N2333/39
摘要： T cell receptors (TCRS) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.
摘要翻译：提供了比野生型TCR对配体具有更高亲和力的T细胞受体（TCRS）。通过诱变T细胞受体蛋白质编码序列产生T细胞受体蛋白编码序列突变体的杂色群体，形成这些高亲和性TCR。将T细胞受体突变体编码序列转化为酵母细胞; 在酵母细胞表面诱导T细胞受体突变体编码序列的表达; 并选择那些表达与野生型T细胞受体蛋白相比对肽/ MHC配体具有更高亲和力的T细胞受体突变体的细胞。高亲和性TCR可用于代替抗体或单链抗体。

10. 发明授权

US06759243B2 High affinity TCR proteins and methods 有权
标题翻译：高亲和力TCR蛋白和方法
公开(公告)号：US06759243B2
公开(公告)日：2004-07-06
申请号：US09731242
申请日：2000-12-06
申请人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
发明人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
IPC分类号： C12N510
CPC分类号： C40B40/02 , C07K14/7051 , C12N15/1037 , G01N33/68 , G01N33/6842 , G01N33/6854 , G01N2333/39
摘要： T cell receptors (TCRs) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.
摘要翻译：提供了比野生型TCR对配体具有更高亲和力的T细胞受体（TCR）。通过诱变T细胞受体蛋白质编码序列产生T细胞受体蛋白编码序列突变体的杂色群体，形成这些高亲和性TCR。将T细胞受体突变体编码序列转化为酵母细胞; 在酵母细胞表面诱导T细胞受体突变体编码序列的表达; 并选择那些表达与野生型T细胞受体蛋白相比对肽/ MHC配体具有更高亲和力的T细胞受体突变体的细胞。高亲和性TCR可用于代替抗体或单链抗体。

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