Abstract
Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call “Epivolve,” that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a “pseudo-hapten” immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.
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References
Fuller EP, O'Neill RJ, Weiner MP (2022) Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution. New Biotechnol 71:1–10. https://doi.org/10.1016/j.nbt.2022.06.003
Holland EG, Buhr DL, Acca FE et al (2013) AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins. J Immunol Methods 394(1-2):55–61. https://doi.org/10.1016/j.jim.2013.05.003
Batonick M, Holland EG, Busygina V et al (2016) Platform for high-throughput antibody selection using synthetically-designed antibody libraries. New Biotechnol 33(5 Pt A):565–573. https://doi.org/10.1016/j.nbt.2015.11.005
Van Deventer JA, Wittrup KD (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol (Clifton, N.J.) 1131:151–181. https://doi.org/10.1007/978-1-62703-992-5_10
Zahnd C, Amstutz P, Plückthun A (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4(3):269–279. https://doi.org/10.1038/nmeth1003
Novotny CP, Lavin K (1971) Some effects of temperature on the growth of F pili. J Bacteriol 107(3):671–682. https://doi.org/10.1128/jb.107.3.671-682.1971
Marks JD, Hoogenboom HR, Bonnert TP et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597. https://doi.org/10.1016/0022-2836(91)90498-u
Hoogenboom HR, Griffiths AD, Johnson KS et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137. https://doi.org/10.1093/nar/19.15.4133
Sheets MD, Amersdorfer P, Finnern R et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci U S A 95(11):6157–6162. https://doi.org/10.1073/pnas.95.11.6157
Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14(3):309–314. https://doi.org/10.1038/nbt0396-309
de Haard HJ, van Neer N, Reurs A et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274(26):18218–18230. https://doi.org/10.1074/jbc.274.26.18218
Haidaris CG, Malone J, Sherrill LA et al (2001) Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods 257(1–2):185–202. https://doi.org/10.1016/s0022-1759(01)00463-x
Knappik A, Ge L, Honegger A et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296(1):57–86. https://doi.org/10.1006/jmbi.1999.3444
Sidhu SS, Li B, Chen Y et al (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
Rauchenberger R, Borges E, Thomassen-Wolf E et al (2003) Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J Biol Chem 278(40):38194–38205. https://doi.org/10.1074/jbc.M303164200
Nelson B, Sidhu SS (2012) Synthetic antibody libraries. Methods Mol Biol (Clifton, N.J.) 899:27–41. https://doi.org/10.1007/978-1-61779-921-1_2
Strachan G, McElhiney J, Drever MR et al (2002) Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli. FEMS Microbiol Lett 210(2):257–261. https://doi.org/10.1111/j.1574-6968.2002.tb11190.x
Zhao Q, Buhr D, Gunter C et al (2018) Rational library design by functional CDR resampling. New Biotechnol 45:89–97. https://doi.org/10.1016/j.nbt.2017.12.005
Kiss MM, Babineau EG, Bonatsakis M et al (2011) Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies. J Immunol Methods 367(1–2):17–26. https://doi.org/10.1016/j.jim.2010.09.034
Nikiforov TT, Rendle RB, Kotewicz ML et al (1994) The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization. PCR Methods Appl 3(5):285–291. https://doi.org/10.1101/gr.3.5.285
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82(2):488–492. https://doi.org/10.1073/pnas.82.2.488
Scholle MD, Kehoe JW, Kay BK (2005) Efficient construction of a large collection of phage-displayed combinatorial peptide libraries. Comb Chem High Throughput Screen 8(6):545–551. https://doi.org/10.2174/1386207054867337
Huang R, Fang P, Kay BK (2012) Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries. Methods (San Diego, Calif.) 58(1):10–17. https://doi.org/10.1016/j.ymeth.2012.08.008
Cadwell RC, Joyce GF (1994) Mutagenic PCR. PCR Methods Appl 3(6):S136–S140. https://doi.org/10.1101/gr.3.6.s136
Jones KS, Chapman AE, Driscoll HA et al (2022) MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot. BioTechniques 72(1):11–20. https://doi.org/10.2144/btn-2021-0078
Ferguson FM, Mendez MQ, Acca EF et al (this volume) Validation and the determination of antibody bioactivity using MILKSHAKE and sundae protocols. In: Phage display: methods and protocols. Springer, New York
Acknowledgments
The authors would like to thank Dr. Brian K. Kay for insightful comments and discussion. This research was funded by NIH Appl. ID 1R43GM146473-01 and NIH Appl. ID 1R44AI177126-01.
Patent protection for the Epivolve technology has been submitted for Abbratech Inc.
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Li, X. et al. (2023). Epivolve: A Protocol for Site-Directed Antibodies. In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_29
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DOI: https://doi.org/10.1007/978-1-0716-3381-6_29
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