In vitro and in vivo evaluation of ⁶⁴Cu-radiolabeled KCCYSL peptides for targeting epidermal growth factor receptor-2 in breast carcinomas

Abstract

Epidermal growth factor receptor-2 (EGFR-2) has been implicated in the pathogenesis of breast and other carcinomas. In this report, we tested the ability of a bacteriophage selected peptide KCCYSL, radiolabeled with ⁶⁴Cu, to image EGFR-2 expressing breast tumors in vivo by positron emission tomography (PET). We evaluated and compared the in vivo tissue distribution and imaging properties of ⁶⁴Cu-X-(Gly-Ser-Gly)-KCCYSL peptide (X = 1,4,7,10, tetraazacyclododecane-N,N',N'',N'''-tetracetic acid, [DOTA] 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid [CB-TE2A], and 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA] chelators) in a human breast cancer xenograft mouse model using dual modality PET and computed tomography (CT). The synthesized peptides DO3A-GSG-KCCYSL, CB-TE2A-GSG-KCCYSL, and NO2A-GSG-KCCYSL were purified, radiolabeled with ⁶⁴Cu, and evaluated for human breast cancer cell (MDA-MB-435) binding, 50% inhibitory concentration, and serum stability. In vivo pharmacokinetic and small animal PET/CT imaging studies were performed using SCID mice bearing MDA-MB-435 xenografts. The radiolabeled peptides bound with an 50% inhibitory concentration of 42.0 ± 10.2 nM (DO3A), 31 ± 7.9 nM (CB-TE2A), and 44.2 ± 6.6 nM (NO2A) to cultured MDA-MB-435 cells. All of the radiolabeled peptides were stable in vitro. The tumor uptake of DO3A, CB-TE2A, and NO2A peptides were 0.73 ± 0.15 percent injected dose per gram (%ID/g), 0.64 ± 0.08%ID/g, and 0.52 ± 0.04%ID/g, respectively at 1 hour. Liver uptake for the ⁶⁴Cu-DO3A-peptide (1.68 ± 0.42%ID/g) was more than that of the ⁶⁴Cu-CB-TE2A-peptide (0.52 ± 0.02% ID/g) and ⁶⁴Cu-NO2A-peptide (0.48 ± 0.05%ID/g) at 2 hours. PET/CT studies revealed successful tumor uptake of ⁶⁴Cu-peptides at 2 hours postinjection. In vivo kidney retention was observed with all of the radiolabeled peptides. The optimization of bifunctional chelators improves the pharmacokinetic properties of the ⁶⁴Cu-labeled GSG-KCCYSL peptide, which enables the selection of a suitable peptide homolog as a PET imaging agent for EGFR-2 expressing breast carcinomas.

FIG. 1.

Chemical structures of DO3A-(GSG)-KCCYSL, CB-TE2A-(GSG)-KCCYSL, and NO2A-(GSG)-KCCYSL.

FIG. 2.

Epidermal growth factor receptor-2 receptor binding properties of the radiolabeled ⁶⁴Cu-DO3A-(GSG)-KCCYSL, ⁶⁴Cu-CB-TE2A-(GSG)-KCCYSL, and ⁴Cu-NO2A-(GSG)-KCCYSL peptides. Approximately, 1.0 × 10⁵ MDA-MB-435 breast carcinoma or normal mammary epithelial cells/tube were incubated at 37°C for different time intervals with 100,000 counts per minute radioligand. The radiolabled peptides bound to human MDA-MB-435 breast carcinoma cells. ⁶⁴Cu-DO3A- peptide (•); ⁶⁴Cu-CB-TE2A-peptide (▪) and ⁶⁴Cu-NO2A- peptide (□).Very minimal or no binding was observed with normal mammary epithelial (184A1) cells using ⁶⁴Cu-DO3A-peptide (▴) or ⁶⁴Cu-CB-TE2A-peptide (♦) or ⁶⁴Cu-NO2A- peptide (○), respectively.

FIG. 3.

Renal blocking studies with bovine serum albumin fragments. Albumin fragments (200 μg) were preinjected in the mice 5 minutes before the injection of radiolabeled peptides (0.185 MBq). The kidney uptake of the radioligands in mice with and without albumin fragments was measured in a γ-counter. Inhibition in the uptake of ⁶⁴Cu-DO3A-(GSG)-KCCYSL (29%, *p = 0.037), ⁶⁴Cu-CB-TE2A-(GSG)-KCCYSL (47%, **p = 0.02), and ⁶⁴Cu-NO2A-(GSG)-KCCYSL (48%, ***p = 0.032) in the presence of albumin fragments are shown.

FIG. 4.

MicroPET/CT of MDA-MB-435 tumor-bearing mice. ⁶⁴Cu-DO3A-(GSG)-KCCYSL, ⁶⁴Cu-CB-TE2A-(GSG)-KCCYSL, or ⁶⁴Cu-NO2A-(GSG)-KCCYSL 12.0 (MBq) each, was injected into the tail vein of SCID mice bearing MDA-MB-435 tumor xenograft. Imaging was acquired 2 hours postinjection, of the radiolabeled peptides in a PET scanner. The PET images were fused with conventional microCT images to validate regions of increased radiolabeled peptide uptake. The figures depict PET/CT images with (A) ⁶⁴Cu-DO3A-(GSG)-KCCYSL; (B) ⁶⁴Cu-CB-TE2A-(GSG)-KCCYSL; and (C) ⁶⁴Cu-NO2A-(GSG)-KCCYSL (t, tumor; l, liver; k, kidney; i, intestine). CT, computed tomography; PET, positron emission tomography.