Abstract
Recent years have seen the use of recombinant proteins in the treatment of different diseases. Among them, monoclonal antibodies (mAbs) are currently the fastest growing class of bio-therapeutic recombinant proteins. Chinese hamster ovary (CHO) cells are the most commonly used host cells for production of these recombinant mAbs. Expression vectors determine the expression level and quality of recombinant mAbs. Currently, few construction strategies for recombinant mAbs expression vectors in CHO cells have been developed, including monocistronic vector, multiple-promoter expression vector, and tricistronic vector mediated by internal ribosome entry site (IRES) or Furin-2A element. Among them, Furin-2A-mediated vector is an effective approach due to advantages of high “self-cleavage” efficiency, and equal expression of light and heavy chains from a single open reading frame. Here, we have reviewed the progress in development of different strategies for constructing recombinant mAb expression vectors in CHO cells and its potential advantages and disadvantages.

References
Ho RJ, Chien J (2014) Trends in translational medicine and drug targeting and delivery: new insights on an old concept-targeted drug delivery with antibody-drug conjugates for cancers. J Pharm Sci 103:71–77. https://doi.org/10.1002/jps.23761
Durocher Y, Butler M (2009) Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20:700–707. https://doi.org/10.1016/j.copbio.2009.10.008
Sun H, Chen Q, Lai H (2017) Development of antibody therapeutics against flaviviruses. Int J Mol Sci 19:54. https://doi.org/10.3390/ijms19010054
Singh S, Kumar N, Dwiwedi P, Charan J, Kaur R, Sidhu P (2017) Monoclonal antibodies: a review. Curr Clin Pharmacol 12:1–15. https://doi.org/10.2174/1574884712666170809124728
Bayat H, Hossienzadeh S, Pourmaleki E, Ahani R, Rahimpour A (2018) Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells. Prep Biochem Biotechnol 48:160–164. https://doi.org/10.1080/10826068.2017.1421966
Mikhail BA, Zang L, Sebastiano R, Santos MR, Bush DR, Karger BL, Ivanov AR (2018) Complementary middle-down and intact monoclonal antibody proteoform characterization bycapillary zone electrophoresis-mass spectrometry. Electrophoresis. https://doi.org/10.1002/elps.201800067
Li CH, Narhi LO, Wen J, Dimitrova M, Wen ZQ, Li J, Pollastrini J, Nguyen X, Tsuruda T, Jiang Y (2012) The Effect of pH, temperature and salt on the stability of E. coli- and CHO -derived IgG1 Fc. Biochemistry 5:10056–10065. https://doi.org/10.1021/bi300702e
Robinson MP, Ke N, Lobstein J, Peterson C, Szkodny A, Mansell TJ, Tuckey C, Riggs PD, Colussi PA, Noren CJ (2015) Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nat Commun 6:8072. https://doi.org/10.1038/ncomms9072
Dalton AC, Barton WA (2014) Over-expression of secreted proteins from mammalian cell lines. Protein Sci 23:517–525. https://doi.org/10.1002/pro.2439
Mohan C, Kim YG, Koo J, Lee GM (2008) Assessment of cell engineering strategies for improved therapeutic protein productioon in CHO cells. Biotechnol J 3:624–630. https://doi.org/10.1002/biot.200700249
Lim Y, Wong NSC, Lee YY, Ku SC, Wong DC, Yap MG (2010) Engineering mammalian cells in bioprocessing-current achievements and future perspectives. Biotechnol Appl Biochem 55:175. https://doi.org/10.1042/BA20090363
Mullard A (2013) 2012 FDA drug approvals. Nat Rev Drug Discov 12:87–90. https://doi.org/10.1038/nrd3946
Mullard A (2012) 2011 FDA drug approvals. Nat Rev Drug Discov 11:91–94. https://doi.org/10.1038/nrd3657
You M, Yang Y, Zhong C, Chen FT, Wang X, Jia TR, Chen YZ, Zhou B, Mi QY, Zhao QJ, An ZQ, Luo EX, SXia N (2018) Efficient mAb production in CHO cells with optimized signal peptide, codon, and UTR. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-8986-5
Jayapal KR, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics from CHO cells-20 years and counting. Chem Eng Prog 103:40–47
Lai T, Yang Y, Ng SK (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel) 6:579–603. https://doi.org/10.3390/ph6050579
Boeger H, Bushnell DA, Davis R, Griesenbeck J, Lorch Y, trattan JS, Westover KD, Kornberg RD (2005) Structural basis of eukaryotic gene transcription. FEBS Lett 579:899–903. https://doi.org/10.1016/j.febslet.2004.11.027
Ho SC, Bardor M, Li B et al (2013) Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells. PLoS ONE 8:e63247. https://doi.org/10.1371/journal.pone.0063247
Rita Costa A, Elisa Rodrigues M, Henriques M, Azeredo J, Oliveira R (2010) Guidelines to cell engineering for monoclonal antibody production. Eur J Pharm Biopharm 74:127–138. https://doi.org/10.1016/j.ejpb.2009.10.002
Wum FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398. https://doi.org/10.1038/nbt1026
Pei Z, Chu L, Zou WG, Qiu S, Qi R, Gu J, Qian C (2004) An oncoliti c adenovrial vector of smac increasee antitumor activity of against HCC in human cells and in mice. Hepatology 39:1371–1381. https://doi.org/10.1002/hep.20203
Abbott WM, Middleton B, Kartberg F, Claesson J, Roth R, Fisher D (2015) Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale. Protein Expr Purif 116:113–119. https://doi.org/10.1016/j.pep.2015.08.016
Bhoskar P, Belongia B, Smith R, Yoon S, Carter T, Xu J (2013) Free light chain content in culture media reflects recombinant monoclonal antibody productivity and quality. Biotechnol Prog 29:1131–1139. https://doi.org/10.1002/btpr.1767
Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC(2005) Onthe optimal ratio of heavy to light chain genes for efficient recombinant antibody productionby CHO cells. Biotechnol Prog 2:122–133. https://doi.org/10.1021/bp049780w
Allera-Moreau C, Chomarat P, Audinot V, Cogé F, Gillard M, Martineau Y, Boutin JA, Prats AC (2006) The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4. Biochimie 88:737–746. https://doi.org/10.1016/j.biochi.2006.05.019
Davies SL, O’Callaghan PM, McLeod J, Pybus LP, Sung YH, Rance J, Wilkinson SJ, Racher AJ, Young RJ, James DC (2011) Impact of gene vector design on the control of recombinant monoclonal antibody production by Chinese hamster ovary cells. Biotechnol Prog 27:1689–1699. https://doi.org/10.1002/btpr.692
Wang W, Jia YL, Li YC, Jing CQ, Guo X, Shang XF, Zhao CP, Wang TY (2017) Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells. Sci Rep 7:10416. https://doi.org/10.1038/s41598-017-10966-y
Ho SC, Bardor M, Feng H, Tong YW, Song Z, Yap MG, Yang Y (2012) IRES-mediated tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157:130–139. https://doi.org/10.1016/j.jbiotec.2011.09.023
Chng J, Wang T, Nian R, .Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y (2015) Cleavage efficient 2A peptides for high level monoclona lantibody expression in CHO cells. MAbs 7:403–412. https://doi.org/10.1080/19420862.2015.1008351
Shatsky IN, Dmitriev SE, Terenin IM, Andreev DE (2010) Cap-and IRES independent scanning mechanism of translation initiation as an alternative to the concept of cellular IRESs. Mol Cells 30:285–293. https://doi.org/10.1007/s10059-010-0149-1
Yamamoto H, Unbehaun A, Spahn CMT (2017) Ribosomal chamber music: toward an understanding of IRES mechanisms. Trends Biochem Sci 42:655–668. https://doi.org/10.1016/j.tibs.2017.06.002
Nakajima K, Ikenaka K, Nakahira K, Morita N, Mikoshiba K (1993) An improved retroviral vector for assaying promoter activity. Analysis of promoter interference in pIP211 vector. FEBS Lett 315:129–133
Barnes LM, Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis ofsuccessful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol Bioeng 96:337–348. https://doi.org/10.1002/bit.21119
Ng SK, Lin W, Sachdeva R, Wang DI, Yap MG (2010) Vector fragmentation: characterizing vector integrity in transfected clones by Southern blotting. Biotechnol Prog 26:11–20. https://doi.org/10.1002/btpr.281
Li JL, Cui WY, Wang XY, Liu J, Hao SJ, Xu J (2017) Construction of internal ribosome entry site-mediated tricistronic vector for expression of monoclonal antibody against human tumor necrosis factor-α in CHO cells (Chinese). Chin J Biol 30:1252–1257
Hennecke M, Kwissa M, Metzger K, Oumard A, Kröger A, Schirmbeck R, Reimann J, Hauser H (2001) Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs. Nucleic Acids Res 29:3327–3334
Doronina VA, de Felipe P, Wu C, Sharma P, Sachs MS et al (2008) Dissection of a co-translational nascent chain separation event. Biochem Soc Trans 36:712–716. https://doi.org/10.1042/BST0360712
Doronina VA, Wu C, de Felipe P, Sachs MS, Ryan MD et al (2008) Site specific release of nascent chains from ribosomes at a sense codon. Mol Cell Biol 28:4227–4239. https://doi.org/10.1128/MCB.00421-08
Donnelly ML et al (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82:1013–1025. https://doi.org/10.1099/0022-1317-82-5-1013
Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, Qian L, Liu J (2017) Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7:2193. https://doi.org/10.1038/s41598-017-02460-2
Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M, Jooss K (2005) Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 23:584–590. https://doi.org/10.1038/nbt1087
Ebadat S, Ahmadi S, Ahmadi M, Nematpour F, Barkhordari F, Mahdian R, Davami F, Mahboudi F (2017) Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells. PLoS ONE 12:e0185967. https://doi.org/10.1371/journal.pone.0185967
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This work was supported by the National Natural Science Foundation of China (No. 81673337), and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 18IRTSTHN027).
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Li, Ym., Tian, Zw., Xu, Dh. et al. Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells. Mol Biol Rep 45, 2907–2912 (2018). https://doi.org/10.1007/s11033-018-4351-0
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DOI: https://doi.org/10.1007/s11033-018-4351-0