Abstract
Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies for a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sidhu SS (2012) Antibodies for all: The case for genome-wide affinity reagents. FEBS Lett 586(17):2778–2779. https://doi.org/10.1016/j.febslet.2012.05.044
Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517):495–497
Tomszak F, Weber S, Zantow J, Schirrmann T, Hust M, Frenzel A (2016) Selection of recombinant human antibodies. Adv Exp Med Biol 917:23–54. https://doi.org/10.1007/978-3-319-32805-8_3
Miersch S, Li Z, Hanna R, McLaughlin ME, Hornsby M, Matsuguchi T, Paduch M, Saaf A, Wells J, Koide S, Kossiakoff A, Sidhu SS (2015) Scalable high throughput selection from phage-displayed synthetic antibody libraries. J Vis Exp 95:51492. https://doi.org/10.3791/51492
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317
Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246(4935):1275–1281
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554. https://doi.org/10.1038/348552a0
Bradbury AR, Sidhu S, Dubel S, McCafferty J (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29(3):245–254. https://doi.org/10.1038/nbt.1791
Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8(7):1177–1194. https://doi.org/10.1080/19420862.2016.1212149
Baker M (2015) Reproducibility crisis: blame it on the antibodies. Nature 521(7552):274–276. https://doi.org/10.1038/521274a
Bradbury A, Pluckthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518(7537):27–29. https://doi.org/10.1038/518027a
Sidhu SS, Fellouse FA (2006) Synthetic therapeutic antibodies. Nat Chem Biol 2(12):682–688. https://doi.org/10.1038/nchembio843
Bond CJ, Wiesmann C, Marsters JC Jr, Sidhu SS (2005) A structure-based database of antibody variable domain diversity. J Mol Biol 348(3):699–709. https://doi.org/10.1016/j.jmb.2005.02.063
Fellouse FA, Li B, Compaan DM, Peden AA, Hymowitz SG, Sidhu SS (2005) Molecular recognition by a binary code. J Mol Biol 348(5):1153–1162. https://doi.org/10.1016/j.jmb.2005.03.041
Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci U S A 101(34):12467–12472. https://doi.org/10.1073/pnas.0401786101
Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (2003) Domain antibodies: proteins for therapy. Trends Biotechnol 21(11):484–490. https://doi.org/10.1016/j.tibtech.2003.08.007
Nilvebrant J, Tessier PM, Sidhu SS (2016) Engineered autonomous human variable domains. Curr Pharm Des 22(43): 6527–6537.
Adams JJ, Nelson B, Sidhu SS (2014) Recombinant genetic libraries and human monoclonal antibodies. Methods Mol Biol 1060:149–170. https://doi.org/10.1007/978-1-62703-586-6_9
Chen G, Sidhu SS (2014) Design and generation of synthetic antibody libraries for phage display. Methods Mol Biol 1131:113–131. https://doi.org/10.1007/978-1-62703-992-5_8
Nelson B, Sidhu SS (2012) Synthetic antibody libraries. Methods Mol Biol 899:27–41. https://doi.org/10.1007/978-1-61779-921-1_2
Fellouse FA, Sidhu S (2013) Making antibodies in bacteria. In: Howard GC, Kase MR (eds) Making and using antibodies: a practical handbook. CRC Press, Boca Raton, FL, pp 151–17222
Barthelemy PA, Raab H, Appleton BA, Bond CJ, Wu P, Wiesmann C, Sidhu SS (2008) Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. J Biol Chem 283(6):3639–3654. https://doi.org/10.1074/jbc.M708536200
Ma X, Barthelemy PA, Rouge L, Wiesmann C, Sidhu SS (2013) Design of synthetic autonomous VH domain libraries and structural analysis of a VH domain bound to vascular endothelial growth factor. J Mol Biol 425(12):2247–2259. https://doi.org/10.1016/j.jmb.2013.03.020
Tonikian R, Sidhu SS (2012) Selecting and purifying autonomous human variable heavy (VH) domains. Methods Mol Biol 911:327–353. https://doi.org/10.1007/978-1-61779-968-6_20
Lee CV, Liang WC, Dennis MS, Eigenbrot C, Sidhu SS, Fuh G (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340(5):1073–1093. https://doi.org/10.1016/j.jmb.2004.05.051
Persson H, Ye W, Wernimont A, Adams JJ, Koide A, Koide S, Lam R, Sidhu SS (2013) CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. J Mol Biol 425(4):803–811. https://doi.org/10.1016/j.jmb.2012.11.037
Sidhu SS, Li B, Chen Y, Fellouse FA, Eigenbrot C, Fuh G (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338(2):299–310. https://doi.org/10.1016/j.jmb.2004.02.050
Ernst A, Avvakumov G, Tong J, Fan Y, Zhao Y, Alberts P, Persaud A, Walker JR, Neculai AM, Neculai D, Vorobyov A, Garg P, Beatty L, Chan PK, Juang YC, Landry MC, Yeh C, Zeqiraj E, Karamboulas K, Allali-Hassani A, Vedadi M, Tyers M, Moffat J, Sicheri F, Pelletier L, Durocher D, Raught B, Rotin D, Yang J, Moran MF, Dhe-Paganon S, Sidhu SS (2013) A strategy for modulation of enzymes in the ubiquitin system. Science 339(6119):590–595. https://doi.org/10.1126/science.1230161
McLaughlin ME, Sidhu SS (2013) Engineering and analysis of peptide-recognition domain specificities by phage display and deep sequencing. Methods Enzymol 523:327–349. https://doi.org/10.1016/B978-0-12-394292-0.00015-1
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Lechner RL, Engler MJ, Richardson CC (1983) Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J Biol Chem 258(18):11174–11184
Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328:333–363
Acknowledgments
Members of the Sidhu lab are acknowledged for input, particularly Alia Pavlenco and Wei Ye. This work was supported by funds from the Swedish Research Council (637-2013-468 to J.N.). We thank Frederic Fellouse for assistance with Fig. 3.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Nilvebrant, J., Sidhu, S.S. (2018). Construction of Synthetic Antibody Phage-Display Libraries. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7447-4_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7446-7
Online ISBN: 978-1-4939-7447-4
eBook Packages: Springer Protocols