Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes

  • Nam-Kyung Lee
  • Scott Bidlingmaier
  • Yang Su
  • Bin Liu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1701)

Abstract

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Key words

Antibody gene diversity library Kappa light chain Lambda light chain ScFv phage display Chain shuffling Antibody affinity maturation Antibody optimization Human monoclonal antibody 

Notes

Acknowledgment

Work in our laboratory is supported by grants from the National Institutes of Health/National Cancer Institute (R01 CA171315, R01 CA118919, and R01 CA129491). NKL received fellowship support from Basic Science Research Program of the National Research Foundation of Korea (NRF) that is funded by the Ministry of Education, Science and Technology (2013R1A6A3A03060495).

References

  1. 1.
    Nelson AL (2010) Antibody fragments: hope and hype. MAbs 2:77–83CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Chan CEZ, Lim APC, MacAry PA et al (2014) The role of phage display in therapeutic antibody discovery. Int Immunol 26:649–657. https://doi.org/10.1093/intimm/dxu082 CrossRefPubMedGoogle Scholar
  3. 3.
    Marks JD, Hoogenboom HR, Bonnert TP et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581–597CrossRefPubMedGoogle Scholar
  4. 4.
    Clackson T, Hoogenboom HR, Griffiths AD et al (1991) Making antibody fragments using phage display libraries. Nature 352:624–628. https://doi.org/10.1038/352624a0 CrossRefPubMedGoogle Scholar
  5. 5.
    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 274:18218–18230CrossRefPubMedGoogle Scholar
  6. 6.
    Barbas CF 3rd, Kang AS, Lerner RA et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978–7982CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hawkins RE, Russell SJ, Winter G (1992) Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J Mol Biol 226:889–896CrossRefPubMedGoogle Scholar
  8. 8.
    Reiter Y, Schuck P, Boyd LF et al (1999) An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface. J Mol Biol 290:685–698. https://doi.org/10.1006/jmbi.1999.2923 CrossRefPubMedGoogle Scholar
  9. 9.
    Lorimer IA, Keppler-Hafkemeyer A, Beers RA et al (1996) Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display. Proc Natl Acad Sci U S A 93:14815–14820CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dooley H, Flajnik MF, Porter AJ (2003) Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 40:25–33CrossRefPubMedGoogle Scholar
  11. 11.
    Hairul Bahara NH, Chin ST, Choong YS et al (2016) Construction of a semisynthetic human VH single-domain antibody library and selection of domain antibodies against alpha-crystalline of mycobacterium tuberculosis. J Biomol Screen 21:35–43. https://doi.org/10.1177/1087057115609144 CrossRefPubMedGoogle Scholar
  12. 12.
    Sanchez-Martin D, Sorensen MD, Lykkemark S et al (2015) Selection strategies for anticancer antibody discovery: searching off the beaten path. Trends Biotechnol 33:292–301. https://doi.org/10.1016/j.tibtech.2015.02.008 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Schwimmer LJ, Huang B, Giang H et al (2013) Discovery of diverse and functional antibodies from large human repertoire antibody libraries. J Immunol Methods 391:60–71. https://doi.org/10.1016/j.jim.2013.02.010 CrossRefPubMedGoogle Scholar
  14. 14.
    Liu B, Marks JD (2000) Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose. Anal Biochem 286:119–128. https://doi.org/10.1006/abio.2000.4788 CrossRefPubMedGoogle Scholar
  15. 15.
    Liu B, Huang L, Sihlbom C et al (2002) Towards proteome-wide production of monoclonal antibody by phage display. J Mol Biol 315:1063–1073. https://doi.org/10.1006/jmbi.2001.5276 CrossRefPubMedGoogle Scholar
  16. 16.
    Liu B, Conrad F, Cooperberg MR et al (2004) Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells. Cancer Res 64:704–710CrossRefPubMedGoogle Scholar
  17. 17.
    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 95:6157–6162CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ruan W, Sassoon A, An F et al (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol Cell Proteomics 5:2364–2373. https://doi.org/10.1074/mcp.M600246-MCP200 CrossRefPubMedGoogle Scholar
  19. 19.
    An F, Drummond DC, Wilson S et al (2008) Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies. Mol Cancer Ther 7:569–578. https://doi.org/10.1158/1535-7163.MCT-07-2132 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Zhu X, Bidlingmaier S, Hashizume R et al (2010) Identification of internalizing human single-chain antibodies targeting brain tumor sphere cells. Mol Cancer Ther 9:2131–2141CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Venet S, Kosco-Vilbois M, Fischer N (2013) Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies. MAbs 5:690–698. https://doi.org/10.4161/mabs.25592 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yip YL, Hawkins NJ, Clark MA et al (1997) Evaluation of different lymphoid tissue sources for the construction of human immunoglobulin gene libraries. Immunotechnology 3:195–203CrossRefPubMedGoogle Scholar
  23. 23.
    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 14:309–314. https://doi.org/10.1038/nbt0396-309 CrossRefPubMedGoogle Scholar
  24. 24.
    Lennard S (2002) Standard protocols for the construction of scFv libraries. Methods Mol Biol 178:59–71PubMedGoogle Scholar
  25. 25.
    Hust M, Dubel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22:8–14. https://doi.org/10.1016/j.tibtech.2003.10.011 CrossRefPubMedGoogle Scholar
  26. 26.
    Welschof M, Terness P, Kipriyanov SM et al (1997) The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody. Proc Natl Acad Sci U S A 94:1902–1907CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hust M, Frenzel A, Meyer T et al (2012) Construction of human naive antibody gene libraries. Methods Mol Biol 907:85–107. https://doi.org/10.1007/978-1-61779-974-7_5 CrossRefPubMedGoogle Scholar
  28. 28.
    Kugler J, Wilke S, Meier D et al (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 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    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 19:4133–4137CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Rogosch T, Kerzel S, Hoi KH et al (2012) Immunoglobulin analysis tool: a novel tool for the analysis of human and mouse heavy and light chain transcripts. Front Immunol 3:176. https://doi.org/10.3389/fimmu.2012.00176 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Glanville J, Zhai W, Berka J et al (2009) Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. Proc Natl Acad Sci U S A. 106: 20216–20221. doi: 10.1073/pnas.0909775106

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Nam-Kyung Lee
    • 1
  • Scott Bidlingmaier
    • 1
  • Yang Su
    • 1
  • Bin Liu
    • 1
  1. 1.Department of Anesthesia, UCSF Helen Diller Family Comprehensive Cancer CenterUniversity of California at San FranciscoSan FranciscoUSA

Personalised recommendations