Influence of chelate conjugation on a newly identified tumor-targeting peptide

The transfer of peptide sequences identified by screening of phage-displayed libraries to clinical application is often difficult. This study investigated whether coupling of a new peptide, FROP-1, to the chelator 1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA) resulted in structural restriction and, consequently, improved binding and stability. Methods: The peptide FROP-1 was coupled to the chelator DOTA and labeled with In-111. The structural changes caused by the addition of the chelator were determined by circular dichroism. The properties of this modified peptide were investigated in in vitro binding assays and monitored for kinetics, competition, and internalization as well as serum stability. A cell-type binding profile was established and the in vivo biodistribution was evaluated in a nude mouse model. Results: When compared with the free peptide without chelator, FROPDOTA revealed different cellular uptake kinetics, reaching a maximum at 2 h in vitro. The cells completely accumulated the tracer, and competition experiments revealed that 99.4% (FRO82-2 cells), 98.6% (MCF-7 cells), or 99.3% (average for 3 primary head and neck tumor cell lines) of tracer accumulation could be suppressed, revealing the specificity of this process. The internalization kinetics determined in MCF-7 cells supported this finding: After an incubation time of 180 min, the major fraction of FROPDOTA was trapped intracellularly. Serum stability experiments revealed an increase in stability due to the chelator, with a half-life of 71 min. Circular dichroism measurements indicated a fixed alpha-helix structure of FROPDOTA representing a strong change in secondary structure. In competition binding experiments, the binding constant (K-D) to FRO82-2 cells was determined to be 494 nM. Despite this avid binding affinity, the binding kinetics were found to be too slow to induce an uptake in vivo before clearance. Consequently, the biodistribution revealed a rapid renal and hepatobiliary clearance, with blood levels dropping from 5.48 +/- 0.26 %lD/g (percentage injected dose per gram) 5 min after injection to 0.77 +/- 0.15 %ID/g at 135 min after injection. Conclusion: This study revealed that peptides that are identified by display techniques may be underrated. Careful alteration of their structure will permit going beyond the possibilities that the limited pool of naturally occurring peptides provide for tumor targeting.