Recently, there has been a lot of interest by using nanobodies (heavy chain-only antibodies produced naturally from the Camelidae) as targeting molecules for molecular imaging, especially for the nuclear medicine imaging. A radiolabeled method that generates a homogeneous product is of utmost importance in radiotracer development for the nuclear medicine imaging. The conventional method for the radiolabeling of nanobodies is non-specifically, which conjugates the radioisotope chelating group to the side chain ɛ-amine group of lysine or sulfhydryl of cysteine of nanobodies, with a shortcoming of produce of the heterogeneous radiotracer. Here we describe a method for the site-specific radioisotope ^99mTc labeling of nanobodies by transpeptidase Sortase A. The radiolabeling process includes two steps: first step, NH₂-GGGGK(HYNIC)-COOH peptide (GGGGK = NH₂-Gly-Gly-Gly-Gly-Lys-COOH, HYNIC = 6-hydrazinonicotinyl) was labeled with ^99mTc to obtain GGGGK-HYNIC-^99mTc; second step, Sortase A catalyzes the formation of a new peptide bond between the peptide motif LPETG (NH₂-Leu-Pro-Glu-Thr-Gly-COOH) expressed C-terminally on the nanobody and the N-terminal of GGGGK-HYNIC-^99mTc. After a simple purification process, homogeneous single-conjugated and stable ^99mTc-labeled nanobodies were obtained in >50% yield. This approach demonstrates that the Sortase A-mediated conjugation is a valuable strategy for the development of site-specifically ^99mTc-labeled nanobodies.

Integrin αvβ6 is expressed at an undetectable level in normal tissues, but is remarkably upregulated during many pathological processes, especially in cancer and fibrosis. Noninvasive imaging of integrin αvβ6 expression using a radiotracer with favorable in vivo pharmacokinetics would facilitate disease diagnosis and therapy monitoring. Through disulfide-cyclized method, we synthesized in this study, a new integrin αvβ6-targeted cyclic peptide (denoted as cHK), and radiolabeled it with^99mTc. The ability of the resulting radiotracer^99mTc–HYNIC–cHK to detect integrin αvβ6 expression in pancreatic cancer xenografts and idiopathic pulmonary fibrosis was evaluated using small-animal single-photon emission computed tomography (SPECT)/computed tomography (CT).^99mTc–HYNIC–cHK showed significantly improved in vivo metabolic stability compared to the linear peptide-based radiotracer^99mTc–HYNIC–HK.^99mTc–HYNIC–cHK exhibited similar biodistribution properties to^99mTc–HYNIC–HK, but the tumor-to-muscle ratio was significantly increased (2.99 ± 0.87 vs. 1.82 ± 0.27, P < 0.05). High-contrast images of integrin αvβ6-positive tumors and bleomycin-induced fibrotic lungs were obtained by SPECT/CT imaging using^99mTc–HYNIC–cHK. Overall, our studies demonstrate that^99mTc–HYNIC–cHK is a promising SPECT radiotracer for the noninvasive imaging of integrin αvβ6 in living subjects.

The reversible combination of a ligand with specific sites on the surface of a receptor is one of the most important processes in biochemistry. A classic equation with a useful simple graphical method was introduced to obtain the equilibrium constant,K_d, and the maximum density of receptors,B_max. The entire¹²⁵I-labeled ligand binding experiment includes three parts: the radiolabeling, cell saturation binding assays and the data analysis. The assay format described here is quick, simple, inexpensive, and effective, and provides a gold standard for the quantification of ligand-receptor interactions. Although the binding assays and quantitative analysis have not changed dramatically compared to the original methods, we integrate all the parts to calculate the parameters in one concise protocol and adjust many details according to our experience. In every step, several optional methods are provided to accommodate different experimental conditions. All these refinements make the whole protocol more understandable and user-friendly. In general, the experiment takes one person less than 8 h to complete, and the data analysis could be accomplished within 2 h.