Abstract
We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99mTc(I)–carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Snider, H. et al. Sentinel node biopsy in the staging of breast cancer. Am. J. Surg . 176, 305–310 ( 1998).
Tilling, R. et al. Technetium-99-Sestamibi scintimammography for the detection of breast carcinoma. J. Nucl. Med. 39, 849– 856 (1998).
Taillefer, R. et al. Myocardial perfusion imaging with 99m-Tc-methoxy-isobutyl-isonitrile (MIBI): comparison of short and long time intervals between rest and stress injections. Preliminary results. Eur. J. Nucl. Med. 13, 515–522 (1988).
Abrams, M.J., Davison, A., Jones, A.G., Costello, C.E., & Pang, H. Synthesis and characterization of hexakis (alkyl-isocyanide) and hexakis (aryl-isocyanide) complexes of technetium (I). Inorg. Chem. 22, 2798–2800 ( 1983).
See protocols for current clinical trials on non-Hodgkins lymphoma with radiolabeled antibodies: http://lymphoma.org/pages/clinlist.html .
Begent, R.H.J. et al. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat. Med. 2, 979–984 (1996).
Plückthun, A. et al. In Antibody engineering: a practical approach. (eds McCafferty, J., Hogenboom, H. & Chiswell, D.) 203–252 (Oxford University Press, Oxford, UK; 1996).
Nedelman, M.A. Rapid infarct imaging with a technetium-99m-labeled antimyosin recombinant single-chain Fv: Evaluation in a canine model of acute myocardial infarction. J. Nucl. Med. 34, 234– 241 (1993).
Liberatore, M. et al. Efficient one-step direct labelling of recombinant antibodies with technetium-99m. Eur. J. Nucl. Med. 22, 1326 –1329 (1995).
Verhaar, M.J. et al. Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine. J. Nucl. Med. 37, 868–872 (1996).
George, A.J.T. et al. Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide. Proc. Natl. Acad. Sci. USA 92, 8358–8362 (1995).
Vallabhajosula, S. et al. Preclinical evaluation of technetium-99m-labeled somatostatin receptor-binding peptides. J. Nucl. Med. 37, 1016–1022 (1996).
Bogdanov, A., Simonova, M. & Weissleder, R. Design of metal-binding green fluorescent protein variants. Biochim. Biophys. Acta 1397, 56– 64 (1998).
Pietersz, G.A., Patrick, M.R. & Chester, K.A. Preclinical characterization and in vivo imaging studies of an engineered recombinant technetium-99m-labeled metallothionein-containing anti-carcinoembryonic antigen single-chain antibody. J. Nucl. Med. 39, 47–56 ( 1998).
Stalteri, M.A., Bansal, S., Hider, R. & Mather, S.J. Comparison of the stability of technetium-labeled peptides to challenge with cysteine. Bioconjugate Chem. 10, 130–136 (1999).
Skerra, A., Pfitzinger, I. & Plückthun, A. The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a general applicable purification technique. Bio/Technology 9, 273– 278 (1991).
Sattelberger, A.P. & Bryan, J.C. In The manganese group (ed. Casey, C.P.) 151–166. (Elsevier Science, New York; 1995).
Spradau, T.W., Edwards, W.B., Anderson, C.J., Welch, M.J. & Katzenellenbogen, J.A. Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyltechnetium-labeled octreotide. Nucl. Med. Biol. 26, 1– 7 (1999).
Alberto, R. et al. A novel organometallic aqua complex of technetium for labeling of biomolecules: synthesis of [99mTc(OH2)3(CO) 3]+ from [99mTcO4]- in aqueous solution and its reaction with a bifunctional ligand. J. Am. Chem. Soc. 120, 7987– 7988 (1998).
Martell, A.E., Smith, R.M. & Motekaitis, R.J. In Critical selected stability constants of metal complexes, NIST Standard Reference Data Base 46, Version 2.0 (NIST, Gaithersburg, MD; 1995).
Adams, G.P. et al. Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies. Cancer Res. 58, 485–490 (1998).
Schott, M.E. et al. Differential metabolic patterns of iodinated versus radiometal chelated anticarcinoma single-chain Fv molecules. Cancer Res. 55, 5323–5329 (1992).
Behr, T.M., Goldenberg, D.M. & Becker, W. Reducing the renal uptake of radiolabeled antibody fragments and peptides for diagnosis and therapy: present status, future prospects and limitations. Eur. J. Nucl. Med. 25, 201– 212 (1998).
Fritzberg, A.R. Advances in 99m-Tc-labeling of antibodies. Nucl. Med. 26, 7–12 (1987).
Alberto, R., Schibli, R., Abram, U., Schubiger, P.A. & Kaden, T.A. Steps towards [(C5Me5)TcO 3]: novel synthesis of [(C5Me5)Tc(CO)3] from [{Tc(μ 3 –OH)(CO)3}4] and oxidation of [(C5Me5)M(CO)3] (M=Tc, Re) with Br. Polyhedron 7, 1079– 1089 (1996).
Knappik, A. & Plückthun, A. Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 8, 81–89 (1995).
Wörn, A. & Plückthun, A. An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly. FEBS Lett. 427, 357–361 (1998).
Vaughan, T.J. et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314 (1996).
Krebber, A. et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35– 55 (1997).
Lindner, P. et al. Specific detection of His-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. Biotechniques 22, 140–149 ( 1997).
Chizzonit, R. et al. Il-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts. J. Immunol. 148, 3117 –3124 (1992).
Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J. & Bunn, P.A. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Methods 72, 77– 89 (1984).
Hynes, N.E., Gerber, H.A., Saurer, S. & Groner, B. Overexpression of the c-erbB-2 protein in human breast tumor cell lines. J. Cell. Biochem. 39, 167–173 ( 1989).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Waibel, R., Alberto, R., Willuda, J. et al. Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)–carbonyl complex. Nat Biotechnol 17, 897–901 (1999). https://doi.org/10.1038/12890
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/12890
This article is cited by
-
Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex
Molecular Imaging and Biology (2021)
-
Imaging using radiolabelled targeted proteins: radioimmunodetection and beyond
EJNMMI Radiopharmacy and Chemistry (2020)
-
Stable salts of the hexacarbonyl chromium(I) cation and its pentacarbonyl-nitrosyl chromium(I) analogue
Nature Communications (2019)
-
Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging
European Journal of Nuclear Medicine and Molecular Imaging (2015)
-
Design and preclinical evaluation of a 99mTc-labelled diabody of mAb J591 for SPECT imaging of prostate-specific membrane antigen (PSMA)
EJNMMI Research (2014)