title: Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.
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.
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel (VUB) , 1090 Brussels, Belgium.
authors.forename: Sam
authors.initials: S
authors.lastname: Massa
authors.forename: Catarina
authors.initials: C
authors.lastname: Xavier
authors.forename: Jens
authors.initials: J
authors.lastname: De Vos
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis; D002162:Camelus / Q000276:immunology*; D003545:Cysteine / Q000737:chemistry* / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D057054:Molecular Imaging; D015335:Molecular Probes / Q000737:chemistry / Q000276:immunology; D009374:Neoplasms, Experimental / Q000175:diagnosis*; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000276:immunology*; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D003545:Cysteine; D015335:Molecular Probes; D018719:Erb-b2 Receptor Tyrosine Kinases; D061905:Single-Domain Antibodies; C508053:ERBB2 protein, human