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

Abstract

Blinatumomab, the bispecific T cell engager antibody (BsAb), 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 vectors 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. Moreover, 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, in to the host genome with no cofactors. In this study, phiC31 integrase technology in combination with DHFR amplification system was used to have an expression vector for future expression of blinatumomab in CHO cells. The gene of interest (BsAb gene) could be joined to DHFR selection marker with the insertion of an internal ribosome entry site (IRES). By positioning the DHFR downstream of BsAb gene and IRES, the transcription of the selection marker can depend on the successful transcription of the BsAb gene, which was located upstream in the expression construct. In this study, FC550A-1 vector was used as the backbone and DHFR selection marker was successfully combined with phiC31 integrase technology to generate a high-expressing construct for BsAb expression in CHO-DG44 cells in future studies.

Keywords: Blinatumomab; CHO cells; DHFR; FC550A-1 vector; phiC31 integrase.

Figure 1

Confirmation of pcDNA 3.1 (+); a) annotated presentation of pcDNA3.1 (+); b) Gel electrophoresis on agarose 1%, lane 1: 1Kb DNA ladder (Fermentas), lane 2: digested pcDNA3.1 (+) plasmid

Figure 2

Amplification of IRES-DHFR gene from pOptiVEC™-TOPO® plasmid and cloning in to pcDNA 3.1(+); a) annotated presentation of pOptiVEC™-TOPO® plasmid; b) Gel electrophoresis on agarose 1% of amplified IRES-DHFR gene, lane 1: blank, lane 2: 1Kb DNA ladder (Fermentas), lane3: amplified IRES-DHFR gene (1100 bp); c) Gel electrophoresis on agarose 1% of digested pcDNA3.1 (+)-IRES-DHFR with XhoI and HindIII restriction enzymes, separately; Lane 1: pcDNA3.1 (+)-IRES-DHFR digested with XhoI (5652 bp and 2389 bp), Lane 2: Digested pcDNA3.1 (+)-IRES-DHFR with HindIII (~8000 bp), lane 3: 1Kb DNA ladder (Fermentas)

Figure 3

Confirmation of FC550A-1- BsAb-IRES-DHFR; a) annotated presentation of FC200A-1 as phiC31 integrase expression vector that was co-transfected with FC550A-1- BsAb-IRES-DHFR; b) annotated presentation of FC550A-1; c) Gel electrophoresis on agarose 1% of digested FC550A-1- BsAb-IRES-DHFR construct with XhoI and SmaI restriction enzymes, lane a: 1Kbp ladder (Fermentas), lane2: digested FC550A-1- BsAb-IRES-DHFR with SmaI resulted in linear construct, lane 3: digested FC550A-1- BsAb- IRES-DHFR with XhoI resulted in three fragments that were 3401 bp, 2385 and 2975 bp

Figure 4

Sequence analysis of FC550A-1- BsAb-IRES-DHFR construct; Gene sequence of BsAb-IRES-DHFR was aligned with the sequence of FC550A-1- BsAb-IRES-DHFR by global alignment of Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi)