Synthesis and Preclinical Evaluation of the Hsp70 Specific Peptide Tracer TPP-PEG24-DFO-[89Zr] for tumor-selective PET imaging

High precision in vivo PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG(24)-DFO[Zr-89] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG(24) moiety supported tracer stability and improved biodistribution characteristics in vivo. The K-d of the tracer ranged in the low nanomolar range (18.9 +/- 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG(24)-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1(+), a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG(24)-DFO[Zr-89] in vivo enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1(+) (6.2 +/- 1.1% ID/g), 4T1 (4.3 +/- 0.7% ID/g), and CT26 (2.6 +/- 0.6% ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 +/- 0.03% ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG(24)-DFO[Zr-89] to detect mHsp70 in vivo suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70.