Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system

Cacciatore, Jonathan J.; Chasin, Lawrence A.; Leonard, Edward F.

doi:10.1016/j.biotechadv.2010.04.003

Cited by 88 publications

(77 citation statements)

References 65 publications

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“…To express the antibody stably and efficiently, we constructed an expression vector that continuously produces high concentrations of the full-length chimeric antibody (19). The vector contains the furin/2A self-processing peptide site (12), a strong promoter CAG, and a DHFR selection and amplification marker.…”

Section: Discussionmentioning

confidence: 99%

A novel anti‐DR5 chimeric antibody and epirubicin synergistically suppress tumor growth

et al. 2012

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SummaryTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells. TRAIL receptor 2 (DR5) expression is high in tumor cells, transformed cells, and clinical tumor specimens and is low in most normal cells and tissues; therefore, DR5 is considered an attractive target for cancer therapy. In this study, HMCAZ5, a novel mousehuman chimeric antibody based on AD5-10, was generated and stably expressed in CHO-dhfr 2 cells. Highly purified HMCAZ5 exhibits a high affinity for the receptor that is equal to the parental mouse antibody, induces apoptosis in various cancer cells but not in normal hepatocytes, and elicits both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in various human cancer cells. The anthracycline anticancer drug epirubicin (EPB) synergizes the cytotoxicity of HMCAZ5 in cancer cells by upregulating DR5 expression on the cell surfaces, enhancing p53 expression, Bid cleavage, and JNK phosphorylation and downregulating c-FLIP expression and Akt phosphorylation. Moreover, HMCAZ5 alone suppresses tumor growth, and EPB augments the tumoricidal activity in human colorectal and hepatocellular tumor xenografts in athymic nude mice. These data suggest that the anti-DR5 chimeric antibody HMCAZ5 may have a clinical use and represents a useful immunological strategy, in combination with chemotherapy, for the treatment of cancer 2012 IUBMB IUBMB Life, 64(9): 757-765, 2012

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Section: Discussionmentioning

confidence: 99%

A novel anti‐DR5 chimeric antibody and epirubicin synergistically suppress tumor growth

et al. 2012

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“…To enhance the efficiency of this system, many researchers have developed novel methods with which highly producing cells can be more stringently selected (Cacciatore et al 2010). A short hairpin RNA (shRNA) vector targeted to the dhfr gene showed a productivity increase in the transgene-amplified stable CHO cells (Wu et al 2008).…”

Section: Discussionmentioning

confidence: 99%

“…However, challenges remain in efforts to accelerate the development of suitable cell lines for producing recombinant proteins and in the isolation of highly producing cells, as this usually requires several months (Cacciatore et al 2010). Dihydrofolate reductase (DHFR) gene amplification, in which a gene of interest is amplified along with a selection marker dhfr gene in the presence of methotrexate (MTX), has been used widely to establish productive cell lines (Omasa 2002).…”

Section: Introductionmentioning

confidence: 99%

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

et al. 2013

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The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.

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“…Varying the types of transcription regulatory elements (e.g., promoter, enhancer, introns, DNA elements) [61 -66] , improving mRNA stability, and modifying translation codons are some commonly used strategies [67] . The gene of interest and selectable marker genes ( dihydrofolate reductase ( DHFR ) or glutamine synthetase ( GS )) are transfected into host cells, either on one or separate plasmids [66,68] . An inhibitor of DHFR or GS enzymes ( methotrexate ( MTX ) or methionine sulfoximine ( MSX )) is used for the selection of transfected cells.…”

Section: Mammalian Cell Technologymentioning

confidence: 99%

“…Attempts are also being made to engineer site -specifi c integration into the host genome for higher heterologous gene expression [69] , increased gene stability, and reduced numbers of clones screened. While biopharmaceutical production in mammalian systems is dominated by CHO cells [66] , several other cells lines are now being explored for production. These cell lines include baby hamster kidney (BHK -21), human embryonic kidney (HEK -293), and mouse myelomas (NS0 and SP2/0).…”

Section: Mammalian Cell Technologymentioning

confidence: 99%

Advances in Protein Production Technologies

Lua

Chuan

2012

Biopharmaceutical Production Technology

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Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system

Cited by 88 publications

References 65 publications

A novel anti‐DR5 chimeric antibody and epirubicin synergistically suppress tumor growth

A novel anti‐DR5 chimeric antibody and epirubicin synergistically suppress tumor growth

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

Advances in Protein Production Technologies

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