Lasso peptide microcin J25 variant containing RGD motif as a PET probe for integrin a v ß 3 in tumor imaging

Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin α_vβ₃ imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [⁶⁴Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t_1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin α_vβ₃ positive U87MG tumor bearing mice, [⁶⁴Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin α_vβ₃. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin α_vβ₃ expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin α_vβ₃ non-specific accumulation of [⁶⁴Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([⁶⁴Cu]Cu-cyclic RGD peptide). These results of the present study indicate that ⁶⁴Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [⁶⁴Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging.