Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging

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

Effect of Dye and Conjugation Chemistry on the Biodistribution Profile of Near-Infrared-Labeled Nanobodies as Tracers for Image-Guided Surgery

Advances in optical imaging technologies have stimulated the development of near-infrared (NIR) fluorescently labeled targeted probes for use in image-guided surgery. As nanobodies have already proven to be excellent candidates for molecular imaging, we aimed in this project to design NIR-conjugated nanobodies targeting the tumor biomarker HER2 for future applications in this field and to evaluate the effect of dye and dye conjugation chemistry on their pharmacokinetics during development. IRDye800CW or IRdye680RD were conjugated either randomly (via lysines) or site-specifically (via C-terminal cysteine) to the anti-HER2 nanobody 2Rs15d. After verification of purity and functionality, the biodistribution and tumor targeting of the NIR-nanobodies were assessed in HER2-positive and -negative xenografted mice. Site-specifically IRDye800CW- and IRdye680RD-labeled 2Rs15d as well as randomly labeled 2Rs15d-IRDye680RD showed rapid tumor accumulation and low nonspecific uptake, resulting in high tumor-to-muscle ratios at early time points (respectively 6.6 ± 1.0, 3.4 ± 1.6, and 3.5 ± 0.9 for HER2-postive tumors at 3 h p.i., while <1.0 for HER2-negative tumors at 3 h p.i., <i>p</i> < 0.05). Contrarily, using the randomly labeled 2Rs15d-IRDye800CW, HER2-positive and -negative tumors could only be distinguished after 24 h due to high nonspecific signals. Moreover, both randomly labeled 2Rs15d nanobodies were not only cleared via the kidneys but also partially via the hepatobiliary route. In conclusion, near-infrared fluorescent labeling of nanobodies allows rapid, specific, and high contrast <i>in vivo</i> tumor imaging. Nevertheless, the fluorescent dye as well as the chosen conjugation strategy can affect the nanobodies’ properties and consequently have a major impact on their pharmacokinetics

Map of Colombia showing the geographic distribution of the major linguistic groups at the time of the Spanish colonization (a) and in the present day (b), the major entrance routes in Colombia during the peopling of South America (dashed arrows in a) and the location of the population groups analysed in this study (c).

<p>Data used to produce this figure are included in references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120155#pone.0120155.ref005" target="_blank">5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120155#pone.0120155.ref009" target="_blank">9</a>].</p

Phylogenetic network of the Native American mtDNA (a) and Y chromosome (b) haplotypes detected in this study.

<p>Circle size is proportional to the number of haplotypes and branch size is proportional to the number of polymorphisms that distinguish each pair of haplotypes. Dashed lines delimit different haplogroups.</p

Radiolabeled Mannosylated Dextran Derivatives Bearing an NIR-Fluorophore for Sentinel Lymph Node Imaging

Current methods for sentinel lymph node (SLN) mapping involve the use of radioactivity detection with technetium-99m sulfur colloid and/or visually guided identification using a blue dye. To overcome the kinetic variations of two individual imaging agents through the lymphatic system, we report herein on two multifunctional macromolecules, <b>5a</b> and <b>6a</b>, that contain a radionuclide (^99mTc or ⁶⁸Ga) and a near-infrared (NIR) reporter for pre- and/or intraoperative SLN mapping by nuclear and NIR optical imaging techniques. Both bimodal probes are dextran-based polymers (10 kDa) functionalized with pyrazole-diamine (Pz) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating units for labeling with <i>fac</i>-[^99mTc­(CO)₃]⁺ or ⁶⁸Ga­(III), respectively, mannose units for receptor targeting, and NIR fluorophore units for optical imaging. The probes allowed a clear visualization of the popliteal node by single-photon emission computed tomography (SPECT/CT) or positron emission tomography (PET/CT), as well as real-time optically guided excision. Biodistribution studies confirmed that both macromolecules present a significant accumulation in the popliteal node (<b>5a</b>: 3.87 ± 0.63% IA/organ; <b>6a</b>: 1.04 ± 0.26% IA/organ), with minimal spread to other organs. The multifunctional nanoplatforms display a popliteal extraction efficiency >90%, highlighting their potential to be further explored as dual imaging agents