Methods and compositions for promoting non-natural amino acid-containing protein production
Inventors
CHEN, Sigeng • Lu, Yingchun • Tian, Feng
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11851662-B2
Publication Date
2023-12-26
Expiration Date
Abstract
Disclosed herein are methods and compositions for generation of cell lines to promote unnatural amino acid-containing protein production using genome engineering technology.
Core Innovation
The disclosure addresses low mammalian yields when genetically incorporating site-specific non-natural amino acids into proteins using an orthogonal aminoacyl tRNA synthetase and an orthogonal suppressor tRNA together with selector codons. It attributes reduced yields to apoptosis induced by excessive uncharged tRNA and stressors during mammalian expression, and therefore focuses on modifying the cell’s apoptotic pathway so that cells tolerate the presence and activity of the orthogonal translation system.
The core approach generates genome-engineered cell lines for incorporating a non-natural amino acid into a protein by inactivating one or more target site(s) or region(s) involved in an apoptotic pathway. In example embodiments, the apoptotic pathway targets relate to Bcl-2 family apoptotic-pathway members, including pro-apoptotic Bcl-2 family members such as BAX and BAK and other Bcl-2 family genes/sites. The disclosure provides that cell lines are provided with a selector codon-containing gene of interest and comprise an orthogonal aminoacyl tRNA synthetase and an orthogonal suppressor tRNA, while nucleic acids selected from SEQ ID NOs: 1 to 6 are introduced to inactivate the selected apoptotic pathway target site(s) or region(s).
The disclosure further supports that combining apoptotic-pathway inactivation with selector-codon based non-natural amino-acid incorporation improves cell viability and increases expression outcomes. It reports that orthogonal tRNA/aminoacyl-tRNA synthetase containing platform cells can show excessive apoptosis, and that CRISPR-mediated inactivation of apoptosis-related targets including BAX and BAK increases viability and reduces apoptotic fractions while increasing titers and specific productivity in batch and fed-batch formats.
Claims Coverage
The independent claims cover a method of generating a cell line for incorporating a non-natural amino acid into a protein by inactivating apoptosis-pathway target site(s) or region(s), together with a selector codon-containing gene of interest and an orthogonal aminoacyl tRNA synthetase and orthogonal suppressor tRNA. The core inventive features are the apoptosis-pathway inactivation and the selector codon translation components.
Apoptotic-pathway site or region inactivation
Inactivating one or more target site(s) or region(s) in a cell or cell line, where the target site(s) or region(s) is involved in an apoptotic pathway, using a nucleic acid selected from SEQ ID NOs: 1 to 6.
Selector codon-containing gene of interest with orthogonal translation components
Providing a cell or cell line expressing a selector codon-containing gene of interest and comprising an orthogonal aminoacyl tRNA synthetase and an orthogonal suppressor tRNA.
Together, the independent claims require engineered cells that combine selector codon-driven incorporation machinery with genome inactivation of apoptotic-pathway target site(s) or region(s) using nucleic acids selected from SEQ ID NOs: 1 to 6, thereby enabling non-natural amino-acid incorporation into proteins in a cell line format.
Stated Advantages
Increased viability and reduced Annexin V-positive apoptotic fractions after apoptosis-pathway inactivation together with orthogonal incorporation components.
Increased titers and increased specific productivity in batch and fed-batch production formats.
Maintained primary sequence of the produced protein after engineering.
Comparable glycoform profiles and aggregation profiles after engineering.
Documented Applications
Production of biotherapeutics including antibodies such as anti-HER2, anti-PSMA, and anti-CD70 using engineered genome-engineered platform or production cell lines that incorporate non-natural amino acids into proteins via selector codons and orthogonal tRNA/RS.
Production of vaccines using the engineered cell line approach for incorporating non-natural amino acids into proteins.
Interested in licensing this patent?