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

Document Type : Research article


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.


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.


Main Subjects

Ecker DM, Jones SD and Levine HL. The therapeutic
monoclonal antibody market. MAbs. (2015): 7:9-14.
Zhu J. Mammalian cell protein expression for
biopharmaceutical production. Biotechnol. Adv.
(2012) 30: 1158-70.
Lifely MR, Hale C, Boyce S, Keen MJ and Phillips J.
Glycosylation and biological activity of CAMPATH1H expressed in different cell lines and grown under
different culture conditions. Glycobiology (1995) 5:
Bendig M. The production of foreign proteins in
mammalian cells. Genet. Eng. (1988) 7: 91-127.
Nott A, Le Hir H and Moore MJ. Splicing enhances
translation in mammalian cells: an additional function
of the exon junction complex. Gene. Dev. (2004) 18:
Keravala A and Calos MP. Site-specific chromosomal
integration mediated by phiC31 integrase. Methods
Mol. Biol. (2008) 435: 165-73.
Kellems RE. Gene amplification in mammalian cells:
strategies for protein production. Curr. Opin. Biotech.
(1991) 2: 723-9.
Agrawal V and Bal M. Strategies for rapid production
of therapeutic proteins in mammalian cells. BioProcess
Int. (2012) 10: 32-48.
Kingston RE, Kaufman RJ, Bebbington C and Rolfe
M. Amplification using CHO cell expression vectors.
Curr. Protocol. Mol. Biolog. (2002) 16: 16.23.
Deer JR and Allison DS. High‐Level Expression of
Proteins in Mammalian Cells Using Transcription
Regulatory Sequences from the Chinese Hamster
EF‐1α Gene. Biotechnol. Prog. (2004) 20: 880-9.
Orlova NA, Kovnir SV, Hodak JA, Vorobiev II,
Gabibov AG and Skryabin KG. Improved elongation
factor-1 alpha-based vectors for stable high-level
expression of heterologous proteins in Chinese
hamster ovary cells. BMC. Biotechnol. (2014) 14: 56.
Spiess C, Zhai Q and Carter PJ. Alternative molecular
formats and therapeutic applications for bispecific
antibodies. Mol. Immunol. (2015) 67: 95-106.
An IRES-based mammalian vector construction
Wu J, Fu J, Zhang M and Liu D. Blinatumomab: a
bispecific T cell engager (BiTE) antibody against
CD19/CD3 for refractory acute lymphoid leukemia. J.
Hematol. Oncol. (2015) 8: 104.
Naddafi F and Davami F. Anti-CD19 monoclonal
antibodies: a new approach to lymphoma therapy. Int.
J. Mol. Cell. Med. (2015) 4: 143.
Buie LW, Pecoraro JJ, Horvat TZ and Daley RJ.
Blinatumomab: a first-in-class bispecific T-cell
engager for precursor B-cell acute lymphoblastic
leukemia. Ann. pharmacother. (2015) 49: 1057-67.
Akbarzadeh-Sharbaf S, Yakhchali B, Minuchehr
Z, Shokrgozar MA and Zeinali S. Expression
enhancement in trastuzumab therapeutic monoclonal
antibody production using genomic amplification with
methotrexate. Avicenna J. Med. Biotechnol. (2013)
5: 87.
Akbarzadeh-Sharbaf S, Yakhchali B, Minuchehr
Z, Shokrgozar MA and Zeinali S. In silico design,
construction and cloning of Trastuzumab humanized
monoclonal antibody: a possible biosimilar for
Herceptin. Adv. Biomed. Res. (2012) 1: 21.
Hennecke M, Kwissa M, Metzger K, Oumard A,
Kröger A, Schirmbeck R, Reimann J and Hauser
H. Composition and arrangement of genes define
the strength of IRES-driven translation in bicistronic
mRNAs. Nucleic. Acids Res. (2001) 29: 3327-34.
Kaufman RJ, Davies MV, Wasley LC and Michnick
D. Improved vectors for stable expression of foreign
genes in mammalian cells by use of the untranslated
leader sequence from EMC virus. Nucleic. Acids Res.
(1991) 19: 4485-90.
Houdebine LM and Attal J. Internal ribosome entry
sites (IRESs): reality and use. Transgenic Res. (1999)
8: 157-77.
Dirks W, Wirth M and Hauser H. Dicistronic
transcription units for gene expression in mammalian
cells. Gene (1993) 128: 247-9.
Zhou Y, Aran J, Gottesman MM and Pastan I.
Co-expression of human adenosine deaminase and
multidrug resistance using a bicistronic retroviral
vector. Hum. Gene Ther. (1998) 9: 287-93.
Bouabe H, Fässler R and Heesemann J. Improvement
of reporter activity by IRES-mediated polycistronic
reporter system. Nucleic Acids Res. (2008) 36: e28.