Identification of a new peptide for fibrosarcoma tumor targeting and imaging in vivo

Abstract

A 12-mer amino acid peptide SATTHYRLQAAN, denominated TK4, was isolated from a phage-display library with fibrosarcoma tumor-binding activity. In vivo biodistribution analysis of TK4-displaying phage showed a significant increased phage titer in implanted tumor up to 10-fold in comparison with normal tissues after systemic administration in mouse. Competition assay confirmed that the binding of TK4-phage to tumor cells depends on the TK4 peptide. Intravenous injection of (131)I-labeled synthetic TK4 peptide in mice showed a tumor retention of 3.3% and 2.7% ID/g at 1- and 4-hour postinjection, respectively. Tumor-to-muscle ratio was 1.1, 5.7, and 3.2 at 1-, 4-, and 24-hour, respectively, and tumors were imaged on a digital γ-camera at 4-hour postinjection. The present data suggest that TK4 holds promise as a lead structure for tumor targeting, and it could be further applied in the development of diagnostic or therapeutic agent.

Figure 1

In vitro binding assay of TK4-phage to NG4TL4-tk tumor cells. (a) NG4TL4-tk cells were incubated with the indicated phages for affinity examination. After extensive washing, bound phages were eluted from cells and titered as pfu. CP1 and CP3 are phages displaying irreverent peptide. M13 is the wild-type phage. (b) Binding of TK4-expressing phage to NG4TL4-tk cells was verified by flow cytometry with an anti-M13 antibody. Untreated cells (cell only), cells treated with 2nd antibodies only, and cells reacted with the wild-type M13 phages (neg phage) served as negative controls. (c) NG4TL4-tk cells were incubated with 5 × 10¹⁰ pfu of TK4 or wild-type (M13) phages. Bound phages were detected using a primary anti-M13 antibody and a FITC-conjugated secondary antibody (green) and were investigated by a fluorescence-activated laser scanning microscope. Nuclei were counterstained with PI solution (red). Wild-type phage and untreated cell served as controls. (d) TK4-displaying phages (10⁷ pfu) were incubated with NG4TL4-tk cell lysate coated in microtiter wells. After washes, the bound phages were detected using an HRP-conjugated anti-M13 antibody and measured at OD 450 nm. BSA and wild-type M13 phage served as control antigen and phage, respectively.

Figure 2

Biodistribution of TK4-displaying phage in vivo. (a) TK4-displaying or CP1 control phage (10¹²) was dissolved in PBS (200 μl) and IV injected into the tumor-bearing mice. The titers of phage (pfu/g of tissue) recovered from tumor and other tissues were averaged from duplications of 3 mice. Error bar represents the Standard Error of Mean (SEM). P-values were calculated by Student's t-Test with two-tails (*, P < .05). (b) TK4-displaying phages were injected into mice in the presence of different amount (0 to 500 μg) of the competitive synthetic TK4 peptide. The phages were eluted form tumor and liver homogenates and titered as pfu.

Figure 3

TK4 peptide-based tumor imaging in vivo. (a) Mice were injected with 50 μg (100 μCi) of [¹³¹I]-FITC-TK4 through tail vein and imaged at indicated time points on (a). Red circles indicate the location of tumor. The injection of ¹³¹I (100 μCi) served as negative control. (b) Mice were injected with [¹³¹I]-FITC-TK4 as described and sacrificed at 4 or 24 hr, from which tumors and muscles were removed, dissected, and imaged using IVIS 200 optical system to detect of FITC signal inside tumor. The injection of ¹³¹I (100 μCi) served as negative control.

Figure 4

Biodistribution of TK4 in tumor-bearing mouse. Animals receiving IV injected ¹³¹I-FITC-TK4 were sacrificed at 1, 4, or 24 hr postinjection. Tumors and other organs were removed and measured for the radioactivity. Results were calculated as percentage of injected dose per gram of tissue (% ID/g). Error bars indicate SEM. N = 5 at each time point.

Figure 5

Inhibitory effect of fibronectin on TK4 binding. (a) *Subject: Novel protein similar to vertebrate fibronectin type III domain-containing protein family [Danio rerio], GenBank: CAQ14006.1. FN3: Fibronectin type III domain. The alignment result was obtained in Protein Blast of NCBI, and edited by NCBI Sequence Viewer (http://www.ncbi.nlm.nih.gov/projects/sviewer/). (b) NG4TL4-tk cells were pretreated with fibronectin in a concentrations of 0 (mock), 2, or 20 μg/ml (30 min, 37°C) before subjected to FITC-TK4 peptide (40 μM) incubation (1 hr, 37°C). Cells were extensively washed with PBS and then fixed in 4% PFA and mounted. Nuclei were counter stained by DAPI. Original magnification × 200.