Experimental study of ¹⁸⁸Re labeled RGD tumor homing peptide modified recombinant human plasminogen kringle5

Objectives As a novel inhibitor of angiogenesis, plasminogen kringle 5 (K5) specially inhibit the proliferation and migration of endothelial cell. To determine whether addition of a tumor-targeting peptide could improve tumor specificity of K5, Here we genetically modified K5 with RGD (Arg-Gly-Asp) motif, labeled K5-RGD with ¹⁸⁸Re in a convenient manner than evaluate its properties in A549 lung adenocarcinoma.

Methods We genetically modified K5 with two RGD (Arg-Gly-Asp) motifs. The fusion protein K5-RGD was expressed in baculovirus vector expression system and the biological activity of K5-RGD was tested by endothelial cell proliferation assay. ¹⁸⁸Re-K5-RGD was obtained by conjugating His group at the C end of K5-RGD with fac-[¹⁸⁸Re(H₂O)₃(CO)₃]⁺. Chelating efficiency of fac-[¹⁸⁸Re(H₂O)₃(CO)₃]⁺ and radiolabeling efficiency of ¹⁸⁸Re-K5-RGD were measured by radio thin-layer chromatography (RTLC). In vitro stability of ¹⁸⁸Re-K5-RGD was determined in human serum at 37°C and analyzed by RTLC. Competition test was also performed to verify the specificity of binding. A biodistribution study was carried out in nude mice bearing A549 lung adenocarcinoma.

Results K5-RGD was expressed at a low level, with a yield of 1.1 mg/L. The labeling efficiency of ¹⁸⁸Re-K5-RGD was 74.5%, radiochemical purity was 97.2% after purification. The affinity of rhk5 was confirmed by competitive binding studies. ¹⁸⁸Re-K5-RGD showed high stability in human serum, the RCP was more than 80% even 12 h after incubation. Competition test showed a high binding specificity. Furthermore, this radio-complex was excreted mainly through kidneys and showed specific tumor uptake in mice bearing A549 tumors.

Conclusions K5-RGD protein was expressed successfully, the purified K5-RGD inhibited the proliferation of endothelial cells. ¹⁸⁸Re-K5-RGD prepared with indirect method had high purity, stability and specificity. Potentially, ¹⁸⁸Re-K5-RGD may be used for tumor imaging.

Research Support National Natural Science Foundation of China (No. 81101071, No. 81071181), Shanghai Leading Academic Discipline Project (S30203), and Shanghai science and technology talent project (No. 11XD1403700).