Preliminary evaluation of 177Lu-labeled knottin peptides for integrin receptor-targeted radionuclide therapy

Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, Lu-177, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts. Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for Lu-177 radiolabeling. The stability of the radiopeptides Lu-177-DOTA-2.5D and Lu-177-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both Lu-177-DOTA-2.5D and Lu-177-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results. DOTA-2.5D and DOTA-2.5F were labeled with Lu-177 at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to Lu-177-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), Lu-177-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of Lu-177-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for Lu-177-DOTA-2.5D and Lu-177-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, Lu-177-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of Lu-177-DOTA-2.5D. Cystine knot peptides can be successfully radiolabeled with Lu-177 for potential therapeutic applications. Knottin 2.5F labeled with Lu-177 exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.