Rapid Development of CHO Cell Lines for High-Level Production of Recombinant Antibodies

Allison, Daniel W.; Brandenstein, Michael; Davis, Lynn; Deer, Jennifer Running; Shah, Nilesh; Ziegler, Kristin

doi:10.12665/j22.allison

Cited by 6 publications

(3 citation statements)

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“…These results are consistent with those of a previous study in which we used CHEF1 vectors to isolate, in 9–12 weeks and without a gene amplification step, CHO cell lines producing an antibody at 15 pg/cell/day (20). Furthermore, one of the cell lines in this study was demonstrated to maintain a constant level of antibody expression after passaging in the absence of methotrexate for >50 generations (20).…”

Section: Discussionsupporting

confidence: 92%

Association of a Toll‐like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic‐refractory Lyme arthritis

Strle¹,

Shin²,

Glickstein³

et al. 2012

Arthritis & Rheumatism

135

132

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Objective Single-nucleotide polymorphisms (SNPs) have been identified in several genes encoding Toll-like receptors (TLRs) that alter immune function, inflammatory responses and disease susceptibility. The SNPs with best evidence for affecting immune function are TLR1 (1805GG), TLR2 (2258GA) and TLR5 (1174CT). Methods We studied the frequency and functional outcome of these polymorphisms in 248 patients with Lyme disease. Cytokine and chemokine levels were determined in serum of patients with erythema migrans (EM), joint fluid of patients with Lyme arthritis, and supernatants of B. burgdorferi-stimulated PBMC from Lyme arthritis patients, using multiplex assays. Results The frequency of TLR1-1805GG polymorphism was greater in patients with antibiotic-refractory arthritis compared with patients with EM or antibiotic-responsive arthritis. Early in the illness, EM patients with 1805GG, primarily those infected with B. burgdorferi RST1 strains, had higher serum levels of IFNγ, CXCL9 and CXCL10, and more severe infection than patients with 1805TG/TT. These inflammatory responses were amplified in patients with Lyme arthritis, and the highest responses were observed in antibiotic-refractory arthritis patients with 1805GG who had been infected with RST1 strains. When PBMC from Lyme arthritis patients were stimulated with a B. burgdorferi RST1 strain, the 1805GG group had significantly larger fold-increase in the levels of IFNγ, CCL2, CXCL9 and CXCL10, than the 1805TG/TT group. In contrast, the TLR2 and TLR5 polymorphisms did not vary among groups in frequency or function. Conclusion The TLR1-1805GG polymorphism in B. burgdorferi RST1-infected patients was associated with stronger TH1-like inflammatory responses, which may set the stage for antibiotic-refractory arthritis.

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Section: Discussionsupporting

confidence: 92%

Association of a Toll‐like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic‐refractory Lyme arthritis

Strle¹,

Shin²,

Glickstein³

et al. 2012

Arthritis & Rheumatism

135

132

View full text Add to dashboard Cite

show abstract

“…These results are consistent with those of a previous study in which we used CHEF1 vectors to isolate, in 9-12 weeks and without a gene amplification step, CHO cell lines producing an antibody at 15 pg/cell/ day (20). Furthermore, one of the cell lines in this study was demonstrated to maintain a constant level of antibody expression after passaging in the absence of methotrexate for >50 generations (20). CHEF1 vectors enabling the highest levels of expression in CHO cells were those that contained at least 4.1 kb of CHEF1 5′ flanking sequence and a 4.2 kb fragment of CHEF1 3′ flanking sequence.…”

Section: Discussionsupporting

confidence: 92%

“…In this report, we describe using CHEF1 vectors to generate moderate to high level expression of six different proteins in CHO cells and at levels in pooled transfectants that were 3-to 26-fold greater than those from two vectors in frequent use for gene expression in mammalian cells. These results are consistent with those of a previous study in which we used CHEF1 vectors to isolate, in 9-12 weeks and without a gene amplification step, CHO cell lines producing an antibody at 15 pg/cell/ day (20). Furthermore, one of the cell lines in this study was demonstrated to maintain a constant level of antibody expression after passaging in the absence of methotrexate for >50 generations (20).…”

Section: Discussionsupporting

confidence: 92%

High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1α Gene

2004

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High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1alpha (EF-1alpha) gene, as well as 12 kb of 5' flanking sequence and 4 kb of 3' flanking sequence. Expression vectors containing 5' and 3' flanking sequences from the Chinese hamster EF-1alpha (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1alpha promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1alpha promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines.

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Large-Scale Exogenous Protein Production in Higher Animal Cells

Whitford¹

2004

The GMO Handbook

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Rapid Development of CHO Cell Lines for High-Level Production of Recombinant Antibodies

Cited by 6 publications

References 0 publications

Association of a Toll‐like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic‐refractory Lyme arthritis

Association of a Toll‐like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic‐refractory Lyme arthritis

High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1α Gene

Large-Scale Exogenous Protein Production in Higher Animal Cells

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