Assessing functional suitability of a lyophilized formulation containing designed ankyrin repeat proteins for radionuclide imaging of HER2/neu overexpression in malignant tumors

Aim. To study in vitro and in vivo the functional suitability of ^99mTc-labeled lyophilized formulation containing designed ankyrin repeat protein (DARPin) G3-(GGGS)₃Cys for radionuclide imaging of HER2/neu overexpression in malignant tumors.
Materials and methods. To create a targeted protein, a modified genetic construct with the sequence encoding DARPin G3-(GGGS)₃Cys was used. To generate the experimental probe, we used a lyophilized formulation containing DARPin G3-(GGGS)₃Cys with auxiliary substances and ^99mTc sodium pertechnetate (500 MBq) incubated at 60 °C for 30 min. Radiochemical purity of ^99mTc-G3-(GGGS)₃Cys was analyzed by thin-layer radiochromatography. SKOV-3, BT-474, and DU-145 cell lines were used to test binding specificity in vitro. The dissociation constant was determined via a saturation binding assay on SKOV-3 cells with a range of protein concentrations from 0.2 to 40 nM. Nu/j mice bearing HER2-positive SKOV-3 xenografts and HER2-negative Ramos xenografts were used to evaluate the targeting properties and biodistribution.
Results. A radiocomplex based on ^99mTc and a lyophilized formulation with DARPin G3-(GGGS)₃Cys was obtained with the radiochemical purity of more than 96%. Binding of ^99mTc-G3-(GGGS)₃Cys to the cells was specific (K_D 3.9 ± 0.5 nM) and proportional to the level of HER2/neu expression in the cells. The uptake of ^99mTc-G3-(GGGS)₃Cys in SKOV-3 xenografts was significantly higher than in Ramos xenografts. ^99mTc-G3-(GGGS)₃Cys demonstrated rapid blood and renal clearance and had low activity in the salivary glands and stomach. Liver uptake was about 5–7%ID/g. In addition, ^99mTc-G3-(GGGS)₃Cys exhibited very low uptakes in the lungs, muscles, small intestine, and bones.
Conclusion. The ^99mTc-labeled lyophilized formulation with DARPin G3-(GGGS)₃Cys is functionally suitable for imaging HER2/neu overexpression in tumors, as it binds specifically to the receptor, is stable in vivo, and has favorable biodistribution in organs and tissues. The radiocomplex based on ^99mTc-G3-(GGGS)₃Cys was obtained by a simple method with high radiochemical purity.

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