Imaging of HER2 Expressing Tumors with ⁶⁸Ga Radiolabeled Peptides

Background: One in eight women will be diagnosed with breast cancer during their lifetime. One type of breast cancer, Human Epidermal Growth Factor Receptor Two (HER2) positive breast cancer is characterized by an upregulation of the HER2 receptor which leads to highly prolific and metastatic nature. The use of molecular imaging probes that can specifically distinguish HER2 positive tumors from HER2 negative tumors, may increase diagnostic accuracy with a full body evaluation without multiple biopsies. Radiolabeled Trastuzumab and Pertuzumab, which are currently in clinical trials for HER2 positive breast cancer imaging, opened the door for imaging of HER2 expression and development of targeted imaging agents. Expanding on this work, radiolabeled peptides were investigated for rapid imaging of HER2 positive lesions.

Methods: Two HER2 specific binding peptides, (P5: (DOTA-Bn-SCN)-PEG2-GSGKCCYSL; P2: DOTA-AHX-LTVYPW), were synthesized for radiolabeling and PET imaging. Each peptide was radiolabeled with ⁶⁸Ga at 95° C while vigorously stirring for 12 minutes and analyzed using high-performance liquid chromatography (HPLC) for radiolabeling efficiency. Radiolabeled peptides were combined with phosphoramidon (PA), a non-specific protease inhibitor, during in vitro and animal studies to slow degradation of each peptide to allow for evaluation of cell specificity and binding. Athymic nude mice were implanted with either HER2 positive BT474 cells or HER2 negative MDA-MB-231 cells. Mice bearing BT474 tumors where previously implanted with a house-made estrogen/cholesterol pellet to aid in tumor growth. Mice were injected with ~100uCi of labeled peptide with or without PA and were imaged at a designated time (15 min, 60min, or 120 min) post-injection. Each mouse was sacrificed and a biodistribution was performed to determine the specific and non-specific uptake of the peptide in each animal.

Results: Radiolabeling for both P5 and P2 was consistently over 95%. The specific activities for each peptide were 2.8 MBq/nmol and 2.2 MBq/nmol respectively. The P5 peptide was chosen to move forward into animal studies. [⁶⁸Ga]P5 showed increased %ID/g in HER2 positive tumors with PA co-injection at all-time points (15 min, 60 min, and 120 min) of: 0.76±0.81, 0.64±0.31, and 0.35±0.10 for non-PA and 1.27±1.34, 1.93±1.16, and 045± 0.73 with PA. The HER2 negative tumors also showed lower values at 60 min and 120 minutes with PA with %ID/g values of 0.69± 1.00 and 0.33± 0.33 respectively.

Conclusions: Preliminary animal studies show that the P5 peptide shows promising in vivo biodistribution. Animal experiments with the P5 peptide are ongoing. The P2 and other peptides will be evaluated for HER2 binding to determine if these alternative peptides will be suitable for animal experiments. Acknowledgements: The authors would like to thank the Lapi Lab and the Cyclotron Crew for their constant support and help. UAB Radiology provided for funding for this research.