Skip to main content
SpringerLink
Log in

Construction of Human Immune and Naive scFv Libraries

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1701))

Abstract

Antibody phage display is the most commonly used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The prerequisite for successful generation of antibodies using phage display is the construction of high-quality antibody gene libraries. Here, we give the detailed methods for the construction of human immune and naive scFv gene libraries.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Reichert JM (2016) Therapeutic monoclonal antibodies approved or in review in the European Union or the United States. In: The Antibody Society. Accessed 24 Jun 2016 http://www.antibodysociety.org/news/approved-antibodies/

  2. Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. MAbs 7:9–14. https://doi.org/10.4161/19420862.2015.989042

    Article  CAS  PubMed  Google Scholar 

  3. Dübel S (2007) Recombinant therapeutic antibodies. Appl Microbiol Biotechnol 74:723–729. https://doi.org/10.1007/s00253-006-0810-y

    Article  PubMed  Google Scholar 

  4. Chatenoud L, Bluestone JA (2007) CD3-specific antibodies: a portal to the treatment of autoimmunity. Nat rev Immunol 7:622–632. Doi: nri2134

    Google Scholar 

  5. Harriman G, Harper LK, Schaible TF (1999) Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58(Suppl 1):I61–I64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dalle S, Thieblemont C, Thomas L, Dumontet C (2008) Monoclonal antibodies in clinical oncology. Anti-cancer agents in medicinal chemistry 8:523–32. Doi: 18537534

    Google Scholar 

  7. Jones SE (2008) Metastatic breast cancer: the treatment challenge. Clin Breast Cancer 8:224–233. doi: R8180138H6520884

    Article  CAS  PubMed  Google Scholar 

  8. Osbourn J, Groves M, Vaughan T (2005) From rodent reagents to human therapeutics using antibody guided selection. Methods 36:61–68. doi: S1046-2023(05)00016-2

    Article  CAS  PubMed  Google Scholar 

  9. Getts DR, Getts MT, McCarthy DP, Chastain EML, Miller SD (2010) Have we overestimated the benefit of human(ized) antibodies? MAbs 2:682–694

    Article  PubMed  PubMed Central  Google Scholar 

  10. Harding FA, Stickler MM, Razo J, DuBridge RB (2010) The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2:256–265

    Article  PubMed  PubMed Central  Google Scholar 

  11. Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8:1177–1194. https://doi.org/10.1080/19420862.2016.1212149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317

    Article  CAS  PubMed  Google Scholar 

  13. Barbas CF, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978–7982. Doi: 1896445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Breitling F, Dübel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104:147–153

    Article  CAS  PubMed  Google Scholar 

  15. Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352:624–628. Doi: 1907718

    Article  CAS  PubMed  Google Scholar 

  16. Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains. Nucleic Acids Res 19:4133–4137. Doi: 1908075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581–597. Doi: 1748994

    Article  CAS  PubMed  Google Scholar 

  18. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554

    Article  CAS  PubMed  Google Scholar 

  19. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Neer NC, Nastri HG, Rondon IJ, Leeds JA, Hufton SE, Huang L, Kashin I, Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, Hoogenboom HR, Ladner RC (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23:344–348. Doi: nbt1067

    Article  CAS  PubMed  Google Scholar 

  20. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (2003) Domain antibodies: proteins for therapy. Trends Biotechnol 21:484–490

    Article  CAS  PubMed  Google Scholar 

  21. Hust M, Dübel S (2005) Phage display vectors for the in vitro generation of human antibody fragments. Methods Mol Biol 295:71–96

    CAS  PubMed  Google Scholar 

  22. Hanes J, Plückthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 94:4937–4942. Doi: 9144168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. He M, Taussig MJ (1997) Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res 25:5132–5134. Doi: 9396828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297–12302. Doi: 9356443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557. Doi: 9181578

    Article  CAS  PubMed  Google Scholar 

  26. King DJ, Bowers PM, Kehry MR, Horlick RA (2014) Mammalian cell display and somatic hypermutation in vitro for human antibody discovery. Curr Drug Discov Technol 11:56–64

    Article  CAS  PubMed  Google Scholar 

  27. Trott M, Weiβ S, Antoni S, Koch J, von Briesen H, Hust M, Dietrich U (2014) Functional characterization of two scFv-fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody. PLoS One 9:e97478. https://doi.org/10.1371/journal.pone.0097478

    Article  PubMed  PubMed Central  Google Scholar 

  28. Arakawa M, Yamashiro T, Uechi G, Tadano M, Nishizono A (2007) Construction of human fab (gamma1/kappa) library and identification of human monoclonal fab possessing neutralizing potency against Japanese encephalitis virus. Microbiol Immunol 51:617–625

    Article  CAS  PubMed  Google Scholar 

  29. Hust M, Dübel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22:8–14

    Article  CAS  PubMed  Google Scholar 

  30. Qi H, Lu H, Qiu H-J, Petrenko V, Liu A (2012) Phagemid vectors for phage display: properties, characteristics and construction. J Mol Biol 417:129–143. https://doi.org/10.1016/j.jmb.2012.01.038

    Article  CAS  PubMed  Google Scholar 

  31. Shirai H, Kidera A, Nakamura H (1999) H3-rules: identification of CDR-H3 structures in antibodies. FEBS Lett 455:188–197. Doi: 10428499

    Article  CAS  PubMed  Google Scholar 

  32. Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A, Kirsch MI, Schütte M, Helmsing S, Meier D, Schirrmann T, Dübel S (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159–170. https://doi.org/10.1016/j.jbiotec.2010.09.945

    Article  CAS  PubMed  Google Scholar 

  33. Johansen LK, Albrechtsen B, Andersen HW, Engberg J (1995) pFab60: a new, efficient vector for expression of antibody fab fragments displayed on phage. Protein Eng 8:1063–1067. Doi: 8771188

    Article  CAS  PubMed  Google Scholar 

  34. Little M, Welschof M, Braunagel M, Hermes I, Christ C, Keller A, Rohrbach P, Kürschner T, Schmidt S, Kleist C, Terness P (1999) Generation of a large complex antibody library from multiple donors. J Immunol Methods 231:3–9. Doi: 10648923

    Article  CAS  PubMed  Google Scholar 

  35. Welschof M, Terness P, Kipriyanov SM, Stanescu D, Breitling F, Dörsam H, Dübel S, Little M, Opelz G (1997) The antigen-binding domain of a human IgG-anti-F(ab’)2 autoantibody. Proc Natl Acad Sci U S A 94:1902–1907. Doi: 9050877

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P, de Bruïne AP, Arends JW, Hoogenboom HR (1999) A large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274:18218–18230. Doi: 10373423

    Article  PubMed  Google Scholar 

  37. McCafferty J, Fitzgerald KJ, Earnshaw J, Chiswell DJ, Link J, Smith R, Kenten J (1994) Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display. Appl Biochem Biotechnol 47:157–171–173. Doi: 7944335

    Article  CAS  PubMed  Google Scholar 

  38. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314. Doi: 9630891

    Article  CAS  PubMed  Google Scholar 

  39. Akamatsu Y, Cole MS, Tso JY, Tsurushita N (1993) Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments. J Immunol 151:4651–4659. Doi: 8409426

    CAS  PubMed  Google Scholar 

  40. Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227:381–388. Doi: 1404359

    Article  CAS  PubMed  Google Scholar 

  41. Nissim A, Hoogenboom HR, Tomlinson IM, Flynn G, Midgley C, Lane D, Winter G (1994) Antibody fragments from a “single pot” phage display library as immunochemical reagents. EMBO J 13:692–698. Doi: 7508862

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Barbas CF, Bain JD, Hoekstra DM, Lerner RA (1992) Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci U S A 89:4457–4461. Doi: 1584777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Desiderio A, Franconi R, Lopez M, Villani ME, Viti F, Chiaraluce R, Consalvi V, Neri D, Benvenuto E (2001) A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold. J Mol Biol 310:603–615. Doi: 11439027

    Article  CAS  PubMed  Google Scholar 

  44. Jirholt P, Ohlin M, Borrebaeck CA, Söderlind E (1998) Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework. Gene 215:471–476. Doi: 9714846

    Article  CAS  PubMed  Google Scholar 

  45. Söderlind E, Strandberg L, Jirholt P, Kobayashi N, Alexeiva V, Aberg AM, Nilsson A, Jansson B, Ohlin M, Wingren C, Danielsson L, Carlsson R, Borrebaeck CA (2000) Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol 18:852–856. Doi: 10932154

    Article  PubMed  Google Scholar 

  46. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wölle J, Plückthun A, Virnekäs B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86. Doi: 10656818

    Article  CAS  PubMed  Google Scholar 

  47. Rothe C, Urlinger S, Löhning C, Prassler J, Stark Y, Jäger U, Hubner B, Bardroff M, Pradel I, Boss M, Bittlingmaier R, Bataa T, Frisch C, Brocks B, Honegger A, Urban M (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376:1182–1200. https://doi.org/10.1016/j.jmb.2007.12.018

    Article  CAS  PubMed  Google Scholar 

  48. Tiller T, Schuster I, Deppe D, Siegers K, Strohner R, Herrmann T, Berenguer M, Poujol D, Stehle J, Stark Y, Heßling M, Daubert D, Felderer K, Kaden S, Kölln J, Enzelberger M, Urlinger S (2013) A fully synthetic human fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5:445–470

    Article  PubMed  PubMed Central  Google Scholar 

  49. Kügler J, Wilke S, Meier D, Tomszak F, Frenzel A, Schirrmann T, Dübel S, Garritsen H, Hock B, Toleikis L, Schütte M, Hust M (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15:10. https://doi.org/10.1186/s12896-015-0125-0

    Article  PubMed  PubMed Central  Google Scholar 

  50. Hust M, Jostock T, Menzel C, Voedisch B, Mohr A, Brenneis M, Kirsch MI, Meier D, Dübel S (2007) Single chain fab (scFab) fragment. BMC Biotechnol 7:14

    Article  PubMed  PubMed Central  Google Scholar 

  51. Steinwand M, Droste P, Frenzel A, Hust M, Dübel S, Schirrmann T (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6:204–218. https://doi.org/10.4161/mabs.27227

    Article  PubMed  Google Scholar 

  52. Rondot S, Koch J, Breitling F, Dübel S (2001) A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19:75–78

    Article  CAS  PubMed  Google Scholar 

  53. Soltes G, Hust M, Ng KKY, Bansal A, Field J, Stewart DIH, Dübel S, Cha S, Wiersma EJ (2007) On the influence of vector design on antibody phage display. J Biotechnol 127:626–637

    Article  CAS  PubMed  Google Scholar 

  54. Pelat T, Hust M, Laffly E, Condemine F, Bottex C, Vidal D, Lefranc M-P, Dübel S, Thullier P (2007) High-affinity, human antibody-like antibody fragment (single-chain variable fragment) neutralizing the lethal factor (LF) of bacillus anthracis by inhibiting protective antigen-LF complex formation. Antimicrob Agents Chemother 51:2758–2764

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kirsch M, Hülseweh B, Nacke C, Rülker T, Schirrmann T, Marschall H-J, Hust M, Dübel S (2008) Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8:66. Doi: 1472-6750-8-66

    Article  PubMed  PubMed Central  Google Scholar 

  56. Frenzel A, Kügler J, Wilke S, Schirrmann T, Hust M (2014) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 1060:215–243. https://doi.org/10.1007/978-1-62703-586-6_12

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This review is an updated and revised version of Ref. 56.

Author information

Authors and Affiliations

  1. YUMAB GmbH, Rebenring 33, 38106, Braunschweig, Germany

    Jonas Kügler & Florian Tomszak

  2. Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    André Frenzel & Michael Hust

  3. YUMAB GmbH, Braunschweig, Germany

    André Frenzel

Authors
  1. Jonas Kügler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Florian Tomszak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André Frenzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Hust
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Hust .

Editor information

Editors and Affiliations

  1. Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kügler, J., Tomszak, F., Frenzel, A., Hust, M. (2018). Construction of Human Immune and Naive scFv 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_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7447-4_1

  • 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

Publish with us

Policies and ethics

Search

Navigation