Enhancement of protein production yield mediated by a fast shuttling CDC42 GTPase
Inventors
Sandig, Volker • Winkler, Karsten • Von Horsten, Henning • Rose, Thomas
Assignees
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Publication Number
US-9163089-B2
Publication Date
2015-10-20
Expiration Date
Abstract
The present invention relates to a cell for producing a secreted protein comprising a polynucleotide comprising a nucleic acid sequence encoding a fast cycling cdc42 mutant and a polynucleotide comprising a nucleic acid sequence encoding a secreted protein. It also relates to a method for producing said cell and to a method for producing a secreted protein using said cell.
Core Innovation
The invention provides a non-adherent cell for producing a secreted protein in which the cell comprises a first polynucleotide encoding a fast cycling cdc42 mutant and a second polynucleotide encoding an exogenous secreted protein. The exogenous secreted protein is an antibody. The use of a self-activating fast-cycling cdc42 mutant is described as increasing secreted-protein yield by accelerating ER-to-Golgi trafficking in non-adherent eukaryotic/vertebrate producer cells.
The fast cycling cdc42 mutant is expressed as a self-activating form rather than a permanently active cdc42 mutant, and variants are described. Specific mutant examples include cdc42F28L, cdc42D118N, and cdc42F28L/D121N, including variants defined by sequence identity criteria relative to a reference SEQ ID.
In some embodiments, production is further supported by co-expressing an adenovirus E1B gene region that includes E1B19K/E1B55K. The disclosed production context includes engineered CHO clones producing increased IgG concentration and improved specific productivity, and it describes enhanced yields under fed-batch conditions with improved viability when E1B is co-expressed.
Claims Coverage
The document includes one independent claim defining a non-adherent producer cell with two polynucleotides: one encoding a fast-cycling cdc42 mutant and one encoding an exogenous secreted antibody. Dependent claims further refine the host-cell embodiment, define specific cdc42 mutant amino-acid substitutions/sequence identity thresholds, and add optional genetic elements and production-workflow constraints.
Fast cycling cdc42 mutant in non-adherent producer cell
A non-adherent cell comprising a first polynucleotide encoding a fast cycling cdc42 mutant.
Exogenous secreted antibody via second polynucleotide
A non-adherent cell comprising a second polynucleotide encoding an exogenous secreted protein, wherein the exogenous secreted protein is an antibody.
Specific cdc42 substitutions relative to SEQ ID NO:8
The first nucleic acid sequence encodes fast-cycling cdc42 mutants with specified amino-acid substitutions at positions relative to SEQ ID NO:8, including non-polar substitution at position 28 (F→non-polar), polar substitution at position 118 (D→polar), or both.
Defined mutant variants by sequence identity threshold
The first nucleic acid sequence encodes cdc42F28L, cdc42D118N, or cdc42F28L/D121N variants with amino-acid sequences matching listed SEQ ID NOs with at least 95% identity.
Vertebrate host-cell embodiment categories
In the vertebrate cell, the mammalian, fish, amphibian, reptilian, and avian cells are limited to specified named species/cell types.
E1B gene region derived from Mastadenovirus genus
The E1B gene region is derived from an Adenovirus of the genus Mastadenovirus.
Allowed order of steps (i), (ii), and (iii)
Steps (i), (ii), and (iii) are performed either in the (i)-(ii)-(iii) order, in the (i)-(iii)-(ii) order, or with step (i) first followed by steps (ii) and (iii) simultaneously.
Overall, the claim coverage centers on a non-adherent cell producing an exogenous secreted antibody by co-introducing a first polynucleotide encoding a fast-cycling cdc42 mutant and a second polynucleotide encoding the antibody, with dependent claims specifying the cdc42 mutant substitution/identity patterns, narrowing vertebrate host-cell categories, optionally incorporating an adenovirus-derived E1B gene region, and constraining the execution order of additional production steps.
Stated Advantages
Increases secreted-protein yield by accelerating ER-to-Golgi trafficking.
Increases IgG concentration and specific productivity in engineered CHO clones.
Improves viability and further enhances yields in fed-batch when E1B co-expression is used.
Documented Applications
Producing secreted proteins, specifically antibodies (e.g., IgG) using non-adherent vertebrate producer cells.
Use in engineered CHO clones and fed-batch culture to increase IgG concentration, specific productivity, and viability.
The partial content references therapeutic antibody examples: Bevacizumab and Trastuzumab.
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