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Selection of Antibody Fragments for CAR-T Cell Therapy from Phage Display Libraries

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Chimeric Antigen Receptor T Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2086))

Abstract

CAR-T cell therapy emerged in the last years as a great promise to cancer treatment. Nowadays, there is a run to improve the breadth of its use, and thus, new chimeric antigen receptors (CAR) are being proposed. The antigen-binding counterpart of CAR is an antibody fragment, scFv (single chain variable fragment), that recognizes a membrane protein associated to a cancer cell. In this chapter, the use of human scFv phage display libraries as a source of new mAbs against surface antigen is discussed. Protocols focusing in the use of extracellular domains of surface protein in biotinylated format are proposed as selection antigen. Elution with unlabeled peptide and selection in solution is described. The analysis of enriched scFvs throughout the selection using NGS is also outlined. Taken together these protocols allow for the isolation of new scFvs able to be useful in the construction of new chimeric antigen receptors for application in cancer therapy.

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References

  1. Maher J, Brentjens RJ, Gunset G et al (2002) Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 receptor. Nat Biotechnol 20:70–75

    Article  CAS  PubMed  Google Scholar 

  2. Brudno JN, Kochenderfer JN (2019) Recent advances in CAR T-cell toxicity: mechanisms, manifestations and management. Blood Rev 34:45–55. https://doi.org/10.1016/J.BLRE.2018.11.002

    Article  CAS  PubMed  Google Scholar 

  3. Srivastava S, Riddell SR (2015) Engineering CAR-T cells: design concepts. Trends Immunol 36:494–502. https://doi.org/10.1016/J.IT.2015.06.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jackson HJ, Rafiq S, Brentjens RJ (2016) Driving CAR T-cells forward. Nat Rev Clin Oncol 13:370–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Smith G (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317. https://doi.org/10.1126/science.4001944

    Article  CAS  PubMed  Google Scholar 

  6. Parmley SF, Smith GP (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305–318. https://doi.org/10.1016/0378-1119(88)90495-7

    Article  CAS  PubMed  Google Scholar 

  7. Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97:391–410. https://doi.org/10.1021/cr960065d

    Article  CAS  PubMed  Google Scholar 

  8. Dantas-Barbosa C, de Macedo Brigido M, Maranhao AQ et al (2012) Antibody phage display libraries: contributions to oncology. Int J Mol Sci 13:5420–5440. https://doi.org/10.3390/ijms13055420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lerner RA (2016) Combinatorial antibody libraries: new advances, new immunological insights. Nat Rev Immunol 16:498–508. https://doi.org/10.1038/nri.2016.67

    Article  CAS  PubMed  Google Scholar 

  10. Hentrich C, Ylera F, Frisch C et al (2018) Monoclonal antibody generation by phage display: history, state-of-the-art, and future. Handb Immunoass Technol:47–80. https://doi.org/10.1016/B978-0-12-811762-0.00003-7

    Chapter  Google Scholar 

  11. Andris-Widhopf J, Steinberger P, Fuller R et al (2001) Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, in: phage display: a laboratory manual, 1st edn. CSHL Press, Cold Spring Harbor, New York

    Google Scholar 

  12. Zhao A, Tohidkia MR, Siegel DL et al (2016) Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol 36:276–289. https://doi.org/10.3109/07388551.2014.958978

    Article  CAS  PubMed  Google Scholar 

  13. Burton DR (2001) Antibody libraries, in: phage display: a laboratory manual, 1st edn. CSHL Press, Cold Spring Harbor, New York

    Google Scholar 

  14. Dantas-Barbosa C, Brígido MM, Maranhão AQ (2005) Construction of a human fab phage display library from antibody repertoires of osteosarcoma patients. Genet Mol Res 4:126–140

    CAS  PubMed  Google Scholar 

  15. Yuan Q-A, Robinson MK, Simmons HH et al (2008) Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning. Cancer Immunol Immunother 57:367–378. https://doi.org/10.1007/s00262-007-0376-2

    Article  CAS  PubMed  Google Scholar 

  16. Giordano RJ, Cardó-Vila M, Lahdenranta J et al (2001) Biopanning and rapid analysis of selective interactive ligands. Nat Med 7:1249–1253. https://doi.org/10.1038/nm1101-1249

    Article  CAS  PubMed  Google Scholar 

  17. Dantas-Barbosa C, Faria FP, Brigido MM et al (2009) Isolation of osteosarcoma-associated human antibodies from a combinatorial fab phage display library. J Biomed Biotechnol 2009:1–8. https://doi.org/10.1155/2009/157531

    Article  CAS  Google Scholar 

  18. Andris-Widhopf J, Steinberger P, Fuller R et al (2011) Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harb Protoc 2011:pdb.prot065573. https://doi.org/10.1101/pdb.prot065573

    Article  PubMed  Google Scholar 

  19. Andris-Widhopf J, Rader C, Steinberger P et al (2000) Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242:159–181. https://doi.org/10.1016/S0022-1759(00)00221-0

    Article  CAS  PubMed  Google Scholar 

  20. Rader C, Steinberger P, Barbas CF III (2001) Selection fron antibody libraries, in: phage display: a laboratory manual, 1st edn. CSHL Press, Cold Spring Harbor, New York

    Google Scholar 

  21. Maranhão AQ, Costa MBW, Guedes L et al (2013) A mouse variable gene fragment binds to DNA independently of the BCR context: a possible role for immature B-cell repertoire establishment. PLoS One 8:e72625. https://doi.org/10.1371/journal.pone.0072625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ravn U, Didelot G, Venet S et al (2013) Deep sequencing of phage display libraries to support antibody discovery. Methods 60:99–110. https://doi.org/10.1016/J.YMETH.2013.03.001

    Article  CAS  PubMed  Google Scholar 

  23. Hemadou A, Giudicelli V, Smith ML et al (2017) Pacific biosciences sequencing and IMGT/HighV-QUEST analysis of full-length single chain fragment variable from an in vivo selected phage-display combinatorial library. Front Immunol 8:1796. https://doi.org/10.3389/fimmu.2017.01796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vaisman-Mentesh A, Wine Y (2018) Monitoring phage biopanning by next-generation sequencing. Methods Mol Biol 1701:463–473. https://doi.org/10.1007/978-1-4939-7447-4_26

    Article  CAS  PubMed  Google Scholar 

  25. Rouet R, Jackson KJL, Langley DB et al (2018) Next-generation sequencing of antibody display repertoires. Front Immunol 9:118. https://doi.org/10.3389/fimmu.2018.00118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sun Y, Sholler GS, Shukla GS et al (2015) Autologous antibodies that bind neuroblastoma cells. J Immunol Methods 426:35–41. https://doi.org/10.1016/J.JIM.2015.07.009

    Article  CAS  PubMed  Google Scholar 

  27. Silacci M, Brack S, Schirru G et al (2005) Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5:2340–2350. https://doi.org/10.1002/pmic.200401273

    Article  CAS  PubMed  Google Scholar 

  28. Webster R (2001) Filamentous phage biology, in: phage display: a laboratory manual, 1st edn. CSHL Press, Cold Spring Harbor, New York

    Google Scholar 

  29. Qi H, Lu H, Qiu H-J et al (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 

  30. 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 19:4133–4137. https://doi.org/10.1093/nar/19.15.4133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Leyton-Castro is a PhD student supported by CAPES scholarship. The authors’ projects have financial support from CNPq, FAP-DF, and BNDES. The authors thank all the funding agencies for their financial support.

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Authors and Affiliations

  1. Molecular Pathology Graduation Programme, School of Medicine, University of Brasilia, Brasilia, Brazil

    Nestor F. Leyton-Castro, Marcelo M. Brigido & Andrea Q. Maranhão

  2. Department of Cell Biology, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil

    Marcelo M. Brigido & Andrea Q. Maranhão

Authors
  1. Nestor F. Leyton-Castro
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  2. Marcelo M. Brigido
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  3. Andrea Q. Maranhão
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Corresponding author

Correspondence to Andrea Q. Maranhão .

Editor information

Editors and Affiliations

  1. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Kamilla Swiech

  2. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Kelen Cristina Ribeiro Malmegrim

  3. Center for Cell-Based Therapy CTC Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Virgínia Picanço-Castro

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Leyton-Castro, N.F., Brigido, M.M., Maranhão, A.Q. (2020). Selection of Antibody Fragments for CAR-T Cell Therapy from Phage Display Libraries. In: Swiech, K., Malmegrim, K., Picanço-Castro, V. (eds) Chimeric Antigen Receptor T Cells. Methods in Molecular Biology, vol 2086. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0146-4_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0146-4_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0145-7

  • Online ISBN: 978-1-0716-0146-4

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