Abstract
Highly functional synthetic antibody libraries can be used to generate antibodies against a multitude of antigens with affinities and specificities that rival or exceed those of natural antibodies. Current design and generation of synthetic antibody libraries are dependent on our insights from previous studies of simplified synthetic antibody libraries, in addition to our knowledge of antibody structure and function and sequence diversity of natural antibody repertoires. We describe a detailed protocol for the design and generation of phage-displayed synthetic antibody libraries built on a single framework with diversity restricted to four complementarity-determining regions by using precisely designed degenerate oligonucleotides. This general methodology could be applied to generation of large, functional synthetic antibody libraries using standard supplies, equipment, and molecular biology techniques.
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References
Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497
Nelson AL, Dhimolea E, Reichert JM (2010) Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9:767–774
Morrow KJ Jr (2012) The new generation of antibody therapeutics: current status and future prospects. Cambridge Healthtech Institute, Needham, MA
Bradbury ARM, Sidhu S, Dübel S et al (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29:245–254
Sidhu SS, Fellouse FA (2006) Synthetic therapeutic antibodies. Nat Chem Biol 2:682–688
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317
Fellouse FA, Sidhu SS (2006) Making antibodies in bacteria. In: Howard GC, Kaser MR (eds) Making and using antibodies: a practical handbook. CRC, Boca Raton, FL, pp 157–180
Johnson G, Wu TT (2000) Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res 28:214–218
Fellouse FA, Li B, Compaan DM et al (2005) Molecular recognition by a binary code. J Mol Biol 348:1153–1162
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 101:12467–12472
Fellouse FA, Esaki K, Birtalan S et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373:924–940
Birtalan S, Zhang Y, Fellouse FA et al (2008) The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies. J Mol Biol 377:1518–1528
Fisher RD, Ultsch M, Lingel A et al (2010) Structure of the complex between HER2 and an antibody paratope formed by side chains from tryptophan and serine. J Mol Biol 402:217–229
Sidhu SS, Li B, Chen Y et al (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338:299–310
Knappik A, Ge LM, 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 296:57–86
Persson H, Ye W, Wernimont A et al (2013) CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. J Mol Biol 425:803–811
Karauzum H, Chen G, Abaandou L et al (2012) Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome. J Biol Chem 287:25203–25215
Koellhoffer JF, Chen G, Sandesara RG et al (2012) Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the Ebola virus envelope glycoprotein. Chembiochem 13:2549–2557
Colwill K, Gräslund S, Persson H et al (2011) A roadmap to generate renewable protein binders to the human proteome. Nat Methods 8:551–561
Laver JD, Ancevicius K, Sollazzo P et al (2012) Synthetic antibodies as tools to probe RNA-binding protein function. Mol Biosyst 8:1650–1657
Qazi O, Rani M, Gnanam AJ et al (2011) Development of reagents and assays for the detection of pathogenic burkholderia species. Faraday Discuss 149:23–36
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Hopp TP, Prickett KS, Price VL et al (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat Biotechnol 6:1204–1210
Lee CV, Sidhu SS, Fuh G (2004) Bivalent antibody phage display mimics natural immunoglobulin. J Immunol Methods 284:119–132
Picken RN, Mazaitis AJ, Maas WK et al (1983) Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli. Infect Immun 42:269–275
Kabat EA, Wu TT, Perry HM et al (1991) Sequences of proteins of immunological interest, 5th edn. National Institutes of Health, Bethesda, MD
Lechner RL, Engler MJ, Richardson CC (1983) Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J Biol Chem 258:11174–11184
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Chen, G., Sidhu, S.S. (2014). Design and Generation of Synthetic Antibody Libraries for Phage Display. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-992-5_8
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DOI: https://doi.org/10.1007/978-1-62703-992-5_8
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Publisher Name: Humana Press, Totowa, NJ
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