Abstract
From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially, the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently, the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL, light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas’ inherent ability to surface display and secrete antibodies, they provide a suitable host for both the selection and the production process, substantially streamlining the process for stable cell line generation, and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398
Walsh G (2010) Post-translational modifications of protein biopharmaceuticals. Drug Discov Today 15:773–780
Jäger V, Büssow K, Wagner A, Weber S, Hust M, Frenzel A, Schirrmann T (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52
Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100:3451–3461
Hacker DL, Balasubramanian S (2017) Recombinant protein production from stable mammalian cell lines and pools. Curr Opin Struct Biol 38:129–136
Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196
Kim JY, Kim YG, Lee GM (2011) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93:917–930
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497
Shulman M, Wilde CD, Köhler G (1978) A better cell line for making hybridomas secreting specific antibodies. Nature 276:269–270
Bradbury A, Plückthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518:27–29
Gaj T, Gersbach CA, Barbas CFIII (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Milcarek C (2011) Hide and go seek: activation of the secretory-specific poly(A) site of IgH by transcription elongation factors. In: Grabowski P (ed), RNA processing, Intech, London, pp 1–35. https://doi.org/10.5772/21186
Pogson M, Parola C, Kelton WJ, Heuberger P, Reddy ST (2016) Immunogenomic engineering of a plug-and-(dis)play hybridoma platform. Nat Commun 7:12535
Reddy ST, Ge X, Miklos AE, Hughes RA, Kang SH, Hoi KH, Chrysostomou C, Hunicke-Smith SP, Iverson BL, Tucker PW, Ellington AD, Georgiou G (2010) Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat Biotechnol 28:957–961
Haessler U, Reddy ST (2014) Using next-generation sequencing for discovery of high-frequency monoclonal antibodies in the variable gene repertoires from immunized mice. Methods Mol Biol 1131:191–203
Fields C, O'Connell D, Xiao S, Lee GU, Billiald P, Muzard J (2013) Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies. Nat Protoc 8:1125–1148
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308
Gibson DC, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
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 monoclonal antibody expression in CHO cells. MAbs 7:403–412
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Parola, C., Mason, D.M., Zingg, A., Neumeier, D., Reddy, S.T. (2018). Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression. In: Hacker, D. (eds) Recombinant Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 1850. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8730-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8730-6_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8729-0
Online ISBN: 978-1-4939-8730-6
eBook Packages: Springer Protocols