Skip to main content
SpringerLink
Log in
Book cover

Synthetic Antibodies pp 145–164Cite as

Utilization of Selenocysteine for Site-Specific Antibody Conjugation

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1575))

Abstract

Site-specific conjugation methods are becoming increasingly important in building next-generation antibody-drug conjugates. We have developed a site-specific conjugation technology based on monoclonal antibodies with engineered selenocysteine (Sec) residues, named selenomabs. Here, we provide protocols for the engineering, expression, and purification of selenomabs in single-chain variable fragment (scFv)-Fc format. Methods for selective conjugation of selenomabs to selenol-reactive compounds and analytical characterization of selenomab conjugates are also included.

This is a preview of subscription content, 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   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Chari RV, Miller ML, Widdison WC (2014) Antibody-drug conjugates: an emerging concept in cancer therapy. Angew Chem Int Ed Engl 53:3796–3827

    Article  CAS  PubMed  Google Scholar 

  2. de Goeij BE, Lambert JM (2016) New developments for antibody-drug conjugate-based therapeutic approaches. Curr Opin Immunol 40:14–23

    Article  PubMed  Google Scholar 

  3. Sassoon I, Blanc V (2013) Antibody-drug conjugate (ADC) clinical pipeline: a review. Methods Mol Biol 1045:1–27

    Article  PubMed  Google Scholar 

  4. Adair JR, Howard PW, Hartley JA, Williams DG, Chester KA (2012) Antibody-drug conjugates—a perfect synergy. Expert Opn Biol Ther 12:1191–1206

    Article  CAS  Google Scholar 

  5. Sievers EL, Senter PD (2013) Antibody-drug conjugates in cancer therapy. Annu Rev Med 64:15–29

    Article  CAS  PubMed  Google Scholar 

  6. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68:9280–9290

    Article  CAS  PubMed  Google Scholar 

  7. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, Chace DF, DeBlanc RL, Gearing RP, Bovee TD, Siegall CB, Francisco JA, Wahl AF, Meyer DL, Senter PD (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778–784

    Article  CAS  PubMed  Google Scholar 

  8. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063–7070

    Article  CAS  PubMed  Google Scholar 

  9. Boswell CA, Mundo EE, Zhang C, Bumbaca D, Valle NR, Kozak KR, Fourie A, Chuh J, Koppada N, Saad O, Gill H, Shen BQ, Rubinfeld B, Tibbitts J, Kaur S, Theil FP, Fielder PJ, Khawli LA, Lin K (2011) Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats. Bioconjug Chem 22:1994–2004

    Article  CAS  PubMed  Google Scholar 

  10. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925–932

    Article  CAS  PubMed  Google Scholar 

  11. Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR (2014) Site-specific antibody drug conjugates for cancer therapy. MAbs 6:34–45

    Article  Google Scholar 

  12. Hofer T, Thomas JD, Burke TR Jr, Rader C (2008) An engineered selenocysteine defines a unique class of antibody derivatives. Proc Natl Acad Sci U S A 105:12451–12456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hofer T, Skeffington LR, Chapman CM, Rader C (2009) Molecularly defined antibody conjugation through a selenocysteine interface. Biochemistry 48:12047–12057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cui H, Thomas JD, Burke TR Jr, Rader C (2012) Chemically programmed bispecific antibodies that recruit and activate T cells. J Biol Chem 287:28206–28214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vire B, Skarzynski M, Thomas JD, Nelson CG, David A, Aue G, Burke TR Jr, Rader C, Wiestner A (2014) Harnessing the fcmu receptor for potent and selective cytotoxic therapy of chronic lymphocytic leukemia. Cancer Res 74:7510–7520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hatfield DL, Gladyshev VN (2002) How selenium has altered our understanding of the genetic code. Mol Cell Biol 22:3565–3576

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, Gladyshev VN (2003) Characterization of mammalian selenoproteomes. Science 300:1439–1443

    Article  CAS  PubMed  Google Scholar 

  18. Labunskyy VM, Hatfield DL, Gladyshev VN (2014) Selenoproteins: molecular pathways and physiological roles. Physiol Rev 94:739–777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li X, Patterson JT, Sarkar M, Pedzisa L, Kodadek T, Roush WR, Rader C (2015) Site-specific dual antibody conjugation via engineered cysteine and selenocysteine residues. Bioconjug Chem 26:2243–2248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Novoselov SV, Lobanov AV, Hua D, Kasaikina MV, Hatfield DL, Gladyshev VN (2007) A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells. Proc Natl Acad Sci U S A 104:7857–7862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yang J, Rader C (2012) Cloning, expression, and purification of monoclonal antibodies in scFv-Fc format. Methods Mol Biol 901:209–232

    Article  CAS  PubMed  Google Scholar 

  22. Pedzisa L, Li X, Rader C, Roush WR (2016) Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts. Org Biomol Chem 14:5141–5147

    Article  CAS  PubMed  Google Scholar 

  23. Patterson JT, Asano S, Li X, Rader C, Barbas CF III. (2014) Improving the serum stability of site-specific antibody conjugates with sulfone linkers. Bioconjug Chem 25:1402–1407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89:4285–4289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way #2C1, Jupiter, FL, 33458, USA

    Xiuling Li & Christoph Rader

  2. Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way #2C1, Jupiter, FL, 33458, USA

    Christoph Rader

Authors
  1. Xiuling Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christoph Rader
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christoph Rader .

Editor information

Editors and Affiliations

  1. MorphoSys AG, Discovery Alliances & Technologies, Planegg, Germany

    Thomas Tiller

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Li, X., Rader, C. (2017). Utilization of Selenocysteine for Site-Specific Antibody Conjugation. In: Tiller, T. (eds) Synthetic Antibodies. Methods in Molecular Biology, vol 1575. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6857-2_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6857-2_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6855-8

  • Online ISBN: 978-1-4939-6857-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation