Using ELP Repeats as a Scaffold for De Novo Construction of Gadolinium-Binding Domains within Multifunctional Recombinant Proteins for Targeted Delivery of Gadolinium to Tumour Cells

Three artificial proteins that bind the gadolinium ion (Gd(3+)) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd(3+)-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd(3+)-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvβ3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.