1. Stable expression of infliximab in CRISPR/Cas9-mediated BAK1-deficient CHO cells.
- Author
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Miao Z, Li Q, Zhao J, Wang P, Wang L, He HP, Wang N, Zhou H, Zhang TC, and Luo XG
- Subjects
- Animals, CHO Cells, CRISPR-Associated Protein 9 metabolism, Clustered Regularly Interspaced Short Palindromic Repeats, Cricetulus, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Gene Editing methods, Gene Expression Profiling, Genomic Instability, Infliximab genetics, Polymerase Chain Reaction, Antirheumatic Agents metabolism, Biotechnology methods, Gene Expression, Infliximab metabolism
- Abstract
Objectives: To establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture., Results: Bcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα)., Conclusions: A CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars.
- Published
- 2018
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