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Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging

Tijdschriftbijdrage - Tijdschriftartikel

Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.
Tijdschrift: Bioconjugate Chemistry
ISSN: 1043-1802
Volume: 25
Pagina's: 979-988
Aantal pagina's: 10
Jaar van publicatie:2014
Trefwoorden:Nanobody, Site-specific labeling, Biochemie & -fysica en Moleculaire biologie, Multidisciplinaire scheikunde , Organische en medische scheikunde
  • Scopus Id: 84900991913
  • ORCID: /0000-0002-4301-5902/work/61226778
  • ORCID: /0000-0001-9006-1074/work/62359703
  • ORCID: /0000-0002-1773-8664/work/62884279
  • ORCID: /0000-0001-9220-4833/work/88545617