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Utilizing A Regulated Targeted Integration Cell Line Development Approach To Systematically Investigate What Makes An Antibody Difficult To Express

T. Tadauchi, Cynthia Lam, Laura Liu, Y. Zhou, D. Tang, S. Louie, B. Snedecor, S. Misaghi
Published 2019 · Biology, Medicine

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Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult‐to‐express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult‐to‐express antibody (mAb‐A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb‐A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb‐A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point‐mutant versions of difficult‐to‐express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.
This paper references
Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells.
G. Ringold (1981)
10.1038/306387A0
Immunoglobulin heavy chain binding protein
Ingrid G. Haas (1983)
10.1128/MCB.4.10.1951
Transfected DNA is mutated in monkey, mouse, and human cells.
J. Lebkowski (1984)
10.1083/JCB.102.5.1558
Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas
D. Bole (1986)
10.1038/NBT0790-662
High Level Expression of Tissue Inhibitor of Metalloproteinases in Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Amplification
M. Cockett (1990)
10.1093/NAR/19.16.4485
Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.
R. Kaufman (1991)
10.1073/PNAS.89.12.5547
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
M. Gossen (1992)
10.1002/j.1460-2075.1992.tb05202.x
Interaction of BiP with newly synthesized immunoglobulin light chain molecules: cycles of sequential binding and release.
M. Knittler (1992)
10.1126/SCIENCE.7792603
Transcriptional activation by tetracyclines in mammalian cells.
M. Gossen (1995)
10.1038/NG0198-56
Repeat-induced gene silencing in mammals
D. Garrick (1998)
10.1016/S0955-0674(02)00324-1
Chromatin as a eukaryotic template of genetic information.
Giacomo Cavalli (2002)
10.1038/nbt1252
Post-translational modifications in the context of therapeutic proteins
G. Walsh (2006)
10.1186/1472-6750-9-100
Recombinant protein expression by targeting pre-selected chromosomal loci
K. Nehlsen (2009)
10.1016/j.copbio.2009.10.008
Expression systems for therapeutic glycoprotein production.
Y. Durocher (2009)
10.1038/nri2761
Therapeutic antibodies for autoimmunity and inflammation
A. Chan (2010)
10.4161/mabs.2.3.11718
Detecting low level sequence variants in recombinant monoclonal antibodies
Yi Yang (2010)
10.1186/1753-6561-5-S8-O6
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
K. Nehlsen (2011)
10.1002/bit.23216
Promoter methylation and transgene copy numbers predict unstable protein production in recombinant chinese hamster ovary cell lines
Andrea Osterlehner (2011)
10.1100/tsw.2011.65
Immunoglobulin Free Light Chain Dimers in Human Diseases
B. Kaplan (2011)
10.1007/s00253-011-3758-5
CHO cells in biotechnology for production of recombinant proteins: current state and further potential
J. Y. Kim (2011)
10.1002/btpr.1783
Fast identification of reliable hosts for targeted cell line development from a limited‐genome screening using combined φC31 integrase and CRE‐Lox technologies
Y. Crawford (2013)
10.3390/ph6050579
Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production
Tingfeng Lai (2013)
10.1002/btpr.1970
It's time to regulate: Coping with product‐induced nongenetic clonal instability in CHO cell lines via regulated protein expression
Shahram Misaghi (2014)
10.1016/j.ymben.2012.12.003
CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.
Valérie Le Fourn (2014)
10.1002/bit.25116
Model‐directed engineering of “difficult‐to‐express” monoclonal antibody production by Chinese hamster ovary cells
Leon P. Pybus (2014)
10.1002/btpr.2175
Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line
L. Zhang (2015)
10.1002/btpr.2439
Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells
Cynthia Lam (2017)
10.1007/978-1-4939-6972-2_1
Strategies and Considerations for Improving Expression of "Difficult to Express" Proteins in CHO Cells.
C. Alves (2017)
10.1002/bit.26268
A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells
Mara C. Inniss (2017)
10.1002/btpr.2611
Investigation of the free heavy chain homodimers of a monoclonal antibody
Hyo (Helen) Chung (2018)
10.1016/j.molcel.2017.06.017
The Unfolded Protein Response and Cell Fate Control.
C. Hetz (2018)



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