Construction of a Mammalian IRES-based Expression Vector to Amplify a Bispecific Antibody; Blinatumomab

Document Type: Research article

Authors

1 Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Biotechnology research center, Pasteur Institute of Iran

3 Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

5 Department of Toxicology, Shadid Beheshti University of Medical Sciences, Tehran, Iran.

Abstract

Blinatumomab, the bispecific T cell engager, has been demonstrated as the most successful BsAb to date. Throughout the past decade, vector design has great importance for the expression of monoclonal antibody in Chinese hamster ovary (CHO) cells. It has been indicated that expression plasmids based on the elongation factor-1 alpha (EF-1 alpha) gene and DHFR selection marker can be highly effective to produce populations of stably transfected cells in the selection medium. Since, the phiC31 integrase system is considered as an attractive and safe protein expression system in mammalian cells and it could integrate a donor plasmid of any size, as a single copy, with no cofactors, we decided to use phiC31 integrase technology in combination with DHFR amplification system to have an expression vector for blinatumomab gene amplification. The gene of interest (GOI) could be joined to DHFR selection marker with the insertion of an internal ribosome entry site (IRES). By positioning the DHFR downstream of the GOI and IRES, the transcription of the selection marker can depend on the successful transcription of the GOI upstream of it in the expression plasmid. In this study, we utilized FC550A-1 vector as the backbone. We successfully combined DHFR selection marker with phiC31 integrase technology to generate a high-expressing plasmid for future expression in CHO-DG44 cells.

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