A TSHR-Targeting Aptamer in Monocytes Correlating with Clinical Activity in TAO.

Abstract

Manifestations of thyroid-associated ophthalmopathy (TAO) vary greatly. Few tools and indicators are available to assess TAO, restricting personalized diagnosis and treatment. To identify an aptamer targeting thyroid-stimulating hormone receptor (TSHR) and utilize this aptamer to evaluate clinical activity in patients with TAO. An aptamer targeting TSHR was developed by exponential enrichment and systematic evaluation of TSHR ligands. After truncation and optimization, the affinity, equilibrium dissociation constant, and serum stability of this aptamer were evaluated. The affinity of the TSHR-targeting aptamer to isolated fibrocytes was assessed, as was aptamer internalization by fibrocytes. The mechanism of binding was determined by molecular docking. The correlation between disease manifestations and the percentage of TSHR-positive cells was assessed by correlation analysis. The aptamer TSHR-21-42 was developed to bind to TSHR, with the equilibrium dissociation constant being 71.46 Kd. Isolated fibrocytes were shown to bind TSHR-21-42 through TSHR, with its affinity maintained at various temperatures and ion concentrations. TSHR-21-42 could compete with anti-TSHR antibody, both for binding site to TSHR and uptake by cells after binding. In addition, TSHR-21-42 could bind to leukocytes in peripheral blood, with this binding differing in patients with TAO and healthy control subjects. The percentage of TSHR-positive monocytes, as determined by binding of TSHR-21-42, correlated positively with clinical activity score in patients with TAO, indicating that TSHR-21-42 binding could assess the severity of TAO. This aptamer targeting TSHR may be used to objectively assess disease activity in patients with TAO, by evaluating the percentages of TSHR positive cells in peripheral blood.