Site-specific labeling of molecular imaging probes allows the development of a homogeneous and well-defined tracer population. The resulting batch-to-batch reproducible pharmacokinetic and -dynamic properties are of great importance for clinical translation. Nanobodies, the recombinantly produced antigen-binding domains of heavy-chain antibodies, have already proven to be proficient probes for non-invasive 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 Nanobodies via a thio-ether bond.
An unpaired cysteine was introduced at the carboxyl-terminal end of the Nanobody 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 hydrochloride 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, and validated in the HER2 cancer model.
After reduction with 2-mercaptoethylamine hydrochloride and conjugation with DTPA-maleimide, a single conjugated tracer population was obtained and confirmed via mass spectrometry. Subsequent labeling with indium-111 resulted in a radiochemical stable complex. The site-specific labeled Nanobody was capable of binding the recombinant HER2 antigen with similar affinity as the parental Nanobody. Moreover, they were able to accumulate significantly in vivo in the tumor of xenografted mice and manifested a high contrast at an early time point after injection (1 h - 1,5 h).
These data provide a versatile and standardized strategy for the site-specific labeling of Nanobodies. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and allows a multimodal alternative to technetium-99m labeling of Nanobodies.
Keywords: homogeneous, nanobody, site-specific labeling
Original languageEnglish
Title of host publicationEMIM Antwerpen
Publication statusPublished - 4 Jun 2014
EventEuropean Molecular Imaging Meeting, EMIM 2014 - Antwerp, Belgium
Duration: 4 Jun 20146 Jun 2014


ConferenceEuropean Molecular Imaging Meeting, EMIM 2014

    Research areas

  • Site-specific labeling, Nanobody

ID: 2475846