Abstract
Antibodies are the fastest growing class of pharmaceutical proteins and essential tools for research and diagnostics. Often antibodies do show a desirable specificity profile but lack sufficient affinity for the desired application. Here, we describe a method to increase the affinity of recombinant antibody fragments based on the construction of mutagenized phage display libraries.
After the construction of a mutated antibody gene library by error-prone PCR, selection of high-affinity variants is either performed by panning in solution or on immobilized antigen with washing conditions optimized for off-rate-dependent selection. An additional screening protocol to identify antibodies with improved thermal stability is described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. mAbs 7(1):9–14
Finlay WJ, Bloom L, Cunningham O (2011) Phage display: a powerful technology for the generation of high specificity affinity reagents from alternative immune sources. Methods Mol Biol 681:87–101
Tiller T et al (2013) A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. mAbs 5(3):445–470
Pantazes RJ, Maranas CD (2013) MAPs: a database of modular antibody parts for predicting tertiary structures and designing affinity matured antibodies. BMC Bioinformatics 14:168
Tomszak F et al (2016) Selection of recombinant human antibodies, in protein targeting compounds. In: Prediction, Selection and Activity of Specific Inhibitors. Springer International Publishing, New York, pp 23–54
McCafferty J (1996) Phage display: factors affecting panning efficiency, in phage display of peptides and proteins. Academic Press, Burlington, pp 261–276
Lamdan H et al (2013) Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor. Mol BioSyst 9(8):2097–2106
Li B et al (2014) In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation. mAbs 6(2):437–445
Douthwaite JA et al (2015) Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class a GPCR formyl-peptide receptor 1. mAbs 7(1):152–166
Rajpal A et al (2005) A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc Natl Acad Sci U S A 102(24):8466–8471
Liu JL et al (2012) Attainment of 15-fold higher affinity of a fusarium-specific single-chain antibody by directed molecular evolution coupled to phage display. Mol Biotechnol 52(2):111–122
Low NM, Holliger P, Winter G (1996) Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mol Biol 260(3):359–368
Chowdhury PS (2002) Targeting random mutations to hotspots in antibody variable domains for affinity improvement. Methods Mol Biol 178:269–285
Laffly E et al (2008) Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six Hypervariable loops. J Mol Biol 378(5):1094–1103
Renaut L et al (2012) Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening. Methods Mol Biol 907:451–461
Hust M et al (2014) Selection of recombinant antibodies from antibody gene libraries. Methods Mol Biol 1101:305–320
Schier R et al (1996) Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J Mol Biol 255(1):28–43
Thie H et al (2011) Rise and fall of an anti-MUC1 specific antibody. PLoS One 6(1):e15921
Friguet B et al (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 77(2):305–319
Della Ducata D et al (2015) Solution equilibrium titration for high-throughput affinity estimation of unpurified antibodies and antibody fragments. J Biomol Screen 20(10):1256–1267
Vernet T et al (2015) Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies. J Mol Recognit 28(10):635–644
Barbas CF III, Burton DR, Scott JK, Silverman GJ (2001) Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p 736
Kügler J et al (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15:10
Welschof M et al (1997) The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody. Proc Natl Acad Sci U S A 94(5):1902–1907
Hust M et al (2007) Handbook of therapeutic antibodies. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Goletz S et al (2002) Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 315(5):1087–1097
Finnern R et al (1997) Human autoimmune anti-proteinase 3 scFv from a phage display library. Clin Exp Immunol 107(2):269–281
Mersmann M et al (1998) Monitoring of scFv selected by phage display using detection of scFv- pIII fusion proteins in a microtiter scale assay. J Immunol Methods 220(1–2):51–58
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
Unkauf, T., Hust, M., Frenzel, A. (2018). Antibody Affinity and Stability Maturation by Error-Prone PCR. 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_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7447-4_22
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