Title | Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging. |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Massa, S., C. Xavier, J. De Vos, V. Caveliers, T. Lahoutte, S. Muyldermans, and N. Devoogdt |
Journal | Bioconjug Chem |
Volume | 25 |
Issue | 5 |
Pagination | 979-88 |
Date Published | 2014 May 21 |
ISSN | 1520-4812 |
Abstract | 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. |
DOI | 10.1021/bc500111t |
Alternate Journal | Bioconjug. Chem. |
PubMed ID | 24815083 |
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