Identification and Evaluation of a New Tumor Cell-Binding Peptide, FROP-1

doi:10.2967/jnumed.106.036699

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Identification and Evaluation of a New Tumor Cell–Binding Peptide, FROP-1

Sabine Zitzmann¹^,2, Susanne Krämer², Walter Mier², Ulrike Hebling¹^,2, Annette Altmann¹^,2, Axel Rother⁴, Dietmar Berndorff⁵, Michael Eisenhut³ and Uwe Haberkorn¹^,2

¹ Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg, Germany; ² Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany; ³ Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Heidelberg, Germany; ⁴ Forschungszentrum Dresden-Rossendorf, Dresden, Germany; and ⁵ Bayer-Schering-Pharma Research Laboratories, Berlin, Germany

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FIGURE 1. In vitro binding studies to various tumor cell lines—MCF-7, MDA-MB435, HeLaTeton, FRO82-2, FTC133, SW1736, PC-3, DU-145, HNO237, HNO97, HNO210, HNO223, CaCo-2, SW948, and HCT116—and to nontumor cell line HPV16-GM and primary endothelial cells HUVEC. Cells were incubated either with ¹²⁵I-FROP-1 without competitor (–) or in presence of 10^–4 M unlabeled FROP-1 (+) for 1 h at 37°C. Experiments were performed in triplicate; SDs are shown.

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FIGURE 2. (A) Amino acid sequence of peptide FROP-1 isolated by peptide phage display. (B) In vitro binding assay with FROP-1. FRO82-2 and MCF-7 cells were grown for 24 h. ¹²⁵I-FROP-1 was added to wells and incubated for 1 h; as competitor, either 1 x 10^–4 M unlabeled FROP-1 or octreotide (Oct) was added immediately before incubation with labeled FROP-1. (C) Internalization of ¹²⁵I-FROP-1 in MCF-7 cells. Cells were incubated with 1–2 x 10⁶ cpm radioligand for 10 min and 1 h at 37°C. After being washed with acidic glycine buffer (pH 2.8), cells were lysed and internalized radioactivity was measured. Experiments were performed in triplicate; SDs are shown. 1W = first acidic wash, 2W = second acidic wash, L = lysate, C = control with unlabeled peptide; Comp. = competitor.

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FIGURE 3. (A and B) In vitro binding kinetics of FROP-1. FRO82-2 and MCF-7 cells were grown for 24 h. ¹²⁵I-FROP-1 was added and incubated for 5, 10, 15, 30, 60, 120, 180, and 240 min without (A) or with (B) 1% BSA. (C) In vitro competition assay with FROP-1 using various competitor concentrations. FRO82-2 and MCF-7 cells were grown for 24 h. Unlabeled FROP-1 in concentrations ranging from 10^–4 M to 10^–7 M were added to cells immediately before ¹²⁵I-FROP-1 and incubated for 1 h. Experiments were performed in triplicate; SDs are shown.

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FIGURE 4. HPLC analysis of ¹²⁵I-FROP-1 in supernatant of FRO82-2 (A) and MCF-7 (B) cells at different time points. Final product of degradation is ¹²⁵I-tyrosine in both cases (peak eluting at approximately 1.55 min).

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FIGURE 5. Stability experiment: ¹²⁵I-FROP-1 was incubated in human serum, and aliquots were removed after times ranging from 5 to 120 min and analyzed by HPLC.

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FIGURE 6. Biodistribution of FROP-1 in female BALB/c nu/nu mice carrying FRO82-2 tumors (n = 3 animals per time point) (A) or MCF-7 tumors (n = 6 animals per time point) (B). Animals received intravenous injection of ¹³¹I-FROP-1, and radioactivity was measured in tumor and control organs after 5, 15, 45, and 135 min. SEM is shown. (C) Tumor-to-muscle ratios calculated for FRO82-2 and MCF-7 tumors at various time points. (D) Biodistribution of ¹²⁵I-FROP-1 at 45 min comparing FRO82-2 and MCF-7 tumors incubated with and without Matrigel (BD Biosciences) (n = 3 animals per time point).