Skip to main content
SpringerLink
Log in
Book cover

Antibody Engineering pp 699–712Cite as

Design and Production of Multimeric Antibody Fragments, Focused on Diabodies with Enhanced Clinical Efficacy

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 907))

Abstract

Multimeric antibody fragments, particularly dimers (diabodies), trimers (triabodies), and tetramers (tetrabodies) of single-chain Fv molecules (scFv), provide high avidity through multivalent binding to the target antigen. The combination of their smaller size and avid binding can provide desirable biological characteristics for tumor targeting applications in vivo; for example, diabodies can have greater tumor penetration and faster blood clearance rates compared to intact full-size antibodies (IgGs). The pharmacokinetic and biodistribution characteristics can further be optimized by the addition of specific thiolation sites for conjugation of PEG molecules to regulate molecular weight and reduce kidney uptake. Thiolation sites can also be used for precise loading of therapeutic payloads. This protocol describes our method for construction and bacterial production of soluble multimeric antibody scFv fragments, focusing on diabodies (scFv dimers).

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   149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
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

Abbreviations

A 600 :

Absorbance at 600 nm

Ab:

Antibody

Amp:

Ampicillin

CDR:

Complementarity determining region

FPLC:

Fast protein liquid chromatography

Fv:

Complex of VH and VL domains

Glu:

Glucose

Ig:

Immunoglobulin

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

kDa:

kiloDalton molecular weight

LDS:

Lithium dodecyl sulfate

MOPS:

3-(N-Morpholino)propanesulfonic acid

MW:

Molecular weight

MWCO:

Molecular weight cutoff

ORF:

Open reading frame

PMSF:

Phenylmethanesulfonyl fluoride

scFv:

Single-chain Fv molecule comprising VH and VL domains joined by a linker

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEC:

Size exclusion chromatography

VH:

Variable domain from antibody heavy chain

VL:

Variable domain from antibody light chain

References

  1. Holliger P, Hudson PJ (2005) Engineered antibodies and the rise of single domains. Nat Biotechnol 23:1126–1136

    Article  CAS  PubMed  Google Scholar 

  2. Kabat EA, Wu TT, Perry HM, Gottensman KS, Foeller C (1991) Sequences of proteins of immunological interest. US Department of Health and Human Service, US Public Health service, NIH, Bethesda, MD

    Google Scholar 

  3. Holliger P, Prospero T, Winter G (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A 90:6444–6448

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Malby R, Caldwell J, Gruen L, Harley V, Ivancic N, Kortt A, Lilley G, Power B, Webster R, Colman P, Hudson P (1993) Recombinant anti-neuraminidase single chain antibody: expression, characterisation and crystallisation in complex with antigen. Proteins 16:57–63

    Article  CAS  PubMed  Google Scholar 

  5. Kortt A, Malby R, Caldwell J, Gruen L, Ivancic N, Lawrence M, Howlett G, Webster R, Hudson P, Colman P (1994) Recombinant anti-neuraminidase single chain Fv antibody: characterization, formation of dimer and higher molecular mass multimers and the solution of the crystal structure of the scFv-neuraminidase complex. Eur J Biochem 221:151–157

    Article  CAS  PubMed  Google Scholar 

  6. Dolezal O, Pearce LA, Lawrence LJ, McCoy AJ, Hudson PJ, Kortt AA (2000) ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass. Protein Eng 13:565–574

    Article  CAS  PubMed  Google Scholar 

  7. Le Gall F, Kipriyanov SM, Moldenhauer G, Little M (1999) Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett 453:164–168

    Article  PubMed  Google Scholar 

  8. Atwell JL, Pearce LA, Lah M, Gruen LC, Kortt AA, Hudson PJ (1996) Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase. Mol Immunol 33:1301–1312

    Article  CAS  PubMed  Google Scholar 

  9. Kipriyanov SM (2009) Generation of bispecific and tandem diabodies. Methods Mol Biol 562:177–193

    Article  CAS  PubMed  Google Scholar 

  10. Hudson PJ, Kortt AA (1999) High avidity ScFv multimers; diabodies and triabodies. J Immunol Methods 231:177–189

    Article  CAS  PubMed  Google Scholar 

  11. Adams GP, Schier R, McCall AM, Crawford RS, Wolf EJ, Weiner LM, Marks JD (1998) Prolonged in vivo tumor retention of a human diabody targeting the extracellular domain of human HERk2/neu. Br J Cancer 77:1405–1412

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Wu AM, Williams LE, Zieran L, Padma A, Sherman M, Bebb GG, Odom-Maryon T, Wong JYC, Shively JE, Raubitschek AA (1999) Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting 4:47–54

    CAS  Google Scholar 

  13. Li L, Crow D, Turatti F, Bading JR, Anderson AL, Poku E, Yazaki PJ, Carmichael J, Leong D, Wheatcroft MP, Raubitschek AA, Hudson PJ, Colcher D, Shively JE (2011) Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing PEG size on kidney clearance and tumor uptake with improved 64-copper PET imaging. Bioconjug Chem 22:709–716

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Sirk SJ, Olafsen T, Barat B, Bauer KB, Wu AM (2008) Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2. Bioconjug Chem 19:2527–2534

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Reddy S, Shaller CC, Doss M, Shchaveleva I, Marks JD, Yu JQ, Robinson MK (2011) Evaluation of the anti-HER2 C6.5 diabody as a PET radiotracer to monitor HER2 status and predict response to trastuzumab treatment. Clin Cancer Res 17:1509–1520

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Olafsen T, Sirk SJ, Betting DJ, Kenanova VE, Bauer KB, Ladno W, Raubitschek AA, Timmerman JM, Wu AM (2010) ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies). Protein Eng Des Sel 23:243–249

    Article  CAS  PubMed  Google Scholar 

  17. Cai W, Olafsen T, Zhang X, Cao Q, Gambhir SS, Williams LE, Wu AM, Chen XJ (2007) PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18 F-labeled T84.66 anti-carcinoembryonic antigen diabody. Nucl Med 48:304–310

    Article  CAS  Google Scholar 

  18. Schneider DW, Heitner T, Alicke B, Light DR, McLean K, Satozawa N, Parry G, Yoo J, Lewis JS, Parry R (2009) In vivo biodistribution, PET imaging, and tumor accumulation of 86Y- and 111In-antimindin/RG-1, engineered antibody fragments in LNCaP tumor-bearing nude mice. J Nucl Med 50:435–443

    Article  CAS  PubMed  Google Scholar 

  19. Robinson MK, Shaller C, Garmestani K, Plascjak PS, Hodge KM, Yuan QA, Marks JD, Waldmann TA, Brechbiel MW, Adams GP (2008) Effective treatment of established human breast tumor xenografts in immunodeficient mice with a single dose of the alpha-emitting radioisotope astatine-211 conjugated to anti-HER2/neu diabodies. Clin Cancer Res 14:875–882

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Barat B, Sirk SJ, McCabe KE, Li J, Lepin EJ, Remenyi R, Koh AL, Olafsen T, Gambhir SS, Weiss S, Wu AM (2009) Cys-diabody quantum dot conjugates (immunoQdots) for cancer marker detection. Bioconjug Chem 20:1474–1481

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Venisnik KM, Olafsen T, Gambhir SS, Wu AM (2007) Fusion of Gaussia luciferase to an engineered anti-carcinoembryonic antigen (CEA) antibody for in vivo optical imaging. Mol Imaging Biol 9:267–277

    Article  PubMed  Google Scholar 

  22. Li L, Turatti F, Crow D, Bading JR, Anderson AL, Poku E, Yazaki PJ, Williams LE, Tamvakis D, Sanders P, Leong D, Raubitschek A, Hudson PJ, Colcher D, Shively JE (2010) Monodispersed DOTA-PEG-conjugated anti-TAG-72 diabody has low kidney uptake and high tumor-to-blood ratios resulting in improved 64Cu PET. J Nucl Med 51:1139–1146

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Müller D, Kontermann RE (2010) The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein. Protein Eng Des Sel 23:827–834

    Article  CAS  PubMed  Google Scholar 

  24. Kenanova VE, Olafsen T, Salazar FB, Williams LE, Knowles S, Wu AM (2010) Tuning the serum persistence of human serum albumin domain III: diabody fusion. Protein Eng Des Sel 23:789–798

    Article  CAS  PubMed  Google Scholar 

  25. Kim KM, McDonagh CF, Westendorf L, Brown LL, Sussman D, Feist T, Lyon R, Alley SC, Okeley NM, Zhang X, Thompson MC, Stone I, Gerber HP, Carter PJ (2008) Anti-CD30 diabody-drug conjugates with potent antitumor activity. Mol Cancer Ther 7:2486–2497

    Article  CAS  PubMed  Google Scholar 

  26. Krauss J, Arndt MA, Vu BK, Newton DL, Seeber S, Rybak SM (2005) Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein. Biochem Biophys Res Commun 331:595–602

    Article  CAS  PubMed  Google Scholar 

  27. Frey K, Schliemann C, Schwager K, Giavazzi R, Johannsen M, Neri D (2010) The immunocytokine F8-IL2 improves the therapeutic performance of sunitinib in a mouse model of renal cell carcinoma. J Urol 184:2540–2548

    Article  CAS  PubMed  Google Scholar 

  28. Nieba L, Honegger A, Krebber C, Plückthun A (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng 10:435–444

    Article  CAS  PubMed  Google Scholar 

  29. Bayly AM, Kortt AA, Hudson PJ, Power BE (2002) Large-scale bacterial fermentation and isolation of scFv multimers using a heat-inducible bacterial expression vector. J Immunol Methods 262:217–227

    Article  CAS  PubMed  Google Scholar 

  30. Yazaki PJ, Shively L, Clark C, Cheung CW, Le W, Szpikowska B, Shively JE, Raubitschek AA, Wu AM (2001) Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications. J Immunol Methods 253:195–208

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Avipep Pty Ltd, Parkville, Australia

    Glenn A. Powers

  2. CSO, Avipep Pty Ltd., Victorian Cancer Diagnostics, Parkville, VIC, Australia

    Peter J. Hudson

  3. Avipep Pty Ltd, Parkville, VIC, Australia

    Michael P. Wheatcroft

Authors
  1. Glenn A. Powers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter J. Hudson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael P. Wheatcroft
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael P. Wheatcroft .

Editor information

Editors and Affiliations

  1. Antibody Therapeutics & Immunotargeting, INSERM U624, Marseille CEDEX 09, France

    Patrick Chames

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Powers, G.A., Hudson, P.J., Wheatcroft, M.P. (2012). Design and Production of Multimeric Antibody Fragments, Focused on Diabodies with Enhanced Clinical Efficacy. In: Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 907. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-974-7_39

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-974-7_39

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-973-0

  • Online ISBN: 978-1-61779-974-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation