In VitroandIn VivoEvaluation of⁶⁴Cu-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 64 Cu, 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 64 Cu-X-(Gly-Ser-Gly)-KCCYSL peptide (X ¼ 1,4,7,10, tetraazacyclododecane-N,N',N'',N'''-tetracetic acid, [DOTA] 1,4,8, 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 64 Cu, 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 AE 10.2 nM (DO3A), 31 AE 7.9 nM (CB-TE2A), and 44.2 AE 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 AE 0.15 percent injected dose per gram (%ID/g), 0.64 AE 0.08%ID/g, and 0.52 AE 0.04%ID/g, respectively at 1 hour. Liver uptake for the 64 Cu-DO3A-peptide (1.68 AE 0.42%ID/g) was more than that of the 64 Cu-CB-TE2A-peptide (0.52 AE 0.02% ID/g) and 64 Cu-NO2A-peptide (0.48 AE 0.05%ID/g) at 2 hours. PET/CT studies revealed successful tumor uptake of 64 Cu-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 64 Cu-labeled GSG-KCCYSL peptide, which enables the selection of a suitable peptide homolog as a PET imaging agent for EGFR-2 expressing breast carcinomas.

Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

“…8,9 The P6.1 peptide, derivatized with different metal chelators (DOTA, NOTA, CB-TE2A, DAP) and labeled with radionuclide metal ions ( 111 In, 64 Cu, 99m Tc), has been successfully used for in vivo imaging of HER-2 overexpressing tumor models. [10][11][12][13] Single-positron emission computed tomography/computed tomography studies showed that radioconjugated peptides were able to accumulate specifically at the tumor site, with K d ranging from 30 to 45 nM, with minimal nonspecific retention in other organs. This peptide sequence has also been proposed for the selective delivery of supramolecular carriers such as copolymeric micelles 14 or pH-tunable liposomes 15 for diagnostic or therapeutic applications.…”

Peptide Phage Display for Discovery of Novel Biomarkers for Imaging and Therapy of Cell Subpopulations in Ovarian Cancer

“…Phage binding affinity may be evaluated by comparing the number of infectious units (TU/mL) between the cancerous and normal cell lines. In order to analyze the binding affinities of peptides outside of the phage environment, biotinylated or radiolabeled peptides may be synthesized and used in in vitro/in vivo binding studies [62,73].…”

“…As described above, selected phage clones may be further analyzed for their binding affinity by micropanning experiments in which phage are incubated with normal ovarian cancer cells and ovarian CSC. Once high binding clones have been identified, biotinylated or radiolabeled peptides can be synthesized and analyzed for their tumor targeting abilities [62,73]. Peptides with high binding affinity for CSC will most likely not be applicable as imaging agents due to the low percentage of CSC in a tumor.…”

“…The a-Melanocyte Stimulating Hocmone (α-MSH) analog has been conjugated with the chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with 64 Cu, 86 Y and 68 Ga for PET imaging of melanoma [97,98]. Our laboratory has identified peptides that bind ovarian, breast, and prostate tumors [59,63,64,73,99,100]. The phage display selected peptide KCCYSL against ErbB-2 has been radiolabeled with 111 In and used for SPECT/ CT imaging of human MDA-MB-435 breast and OV-CAR-3 and SK-OV-3 ovarian xenografted tumors (Figure 2) [58,59,73,99].…”

Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor

“…For the present studies, L19K-FDNB was modified to incorporate 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) at the peptide's N terminus (NO2A). We radiolabeled the peptide with 64 Cu ( 64 Cu half-life 5 12.7 h; b 1 5 17.8%, E max 5 653 keV; b 2 5 38.4%, E max 5 578 keV), because 64 Cu-NOTA complexes are stable in vitro and in vivo (18)(19)(20). We hypothesized the radiolabeled irreversible peptide agent 64 Cu-L19K-FDNB would improve tumor uptake, compared with its reversibly binding peptide analogs 64 Cu-L19K-(2,4-dinitrophenyl) (64Cu-L19K-DNP) and 64 Cu-L19K.…”