Synthesis and Evaluation of 18F-Labeled GRP78-Targeting Probe for Prostate Cancer Imaging with PET

Purpose: The glucose-regulated protein 78K (GRP78) overexpression is closely associated with aggressive growth and invasive properties of many human cancers. In this study, we developed a ¹⁸F labeled BC71 peptide for positron emission tomography (PET) imaging of prostate cancer by targeting GRP78 overexpression.

Methods: GRP78-targeting peptide BC71 was first conjugated with a macrocyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the resulted bioconjugate NOTA-BC71 was then ¹⁸F-labeled to synthesize ¹⁸F-AlF-NOTA-BC71. The disassociation constant of ¹⁸F-AlF-NOTA-BC71 for PC3 tumor cell line was measured through Scatchard plot. The tumor targeting efficacy and in vivo profile of ¹⁸F-AlF-NOTA-BC71 were further evaluated in a subcutaneous PC3 prostate cancer xenograft model by PET and biodistribution study.

Results: NOTA-BC71 was successfully ¹⁸F-fluorinated with good yield within 40 min using the Al¹⁸F intermediate. The K_d of ¹⁸F-AlF-NOTA-BC71 was determined to be 3.46 ± 0.48 nM by Saturation binding assay. Serum stability of ¹⁸F-AlF-NOTA-BC71 demonstrated its excellent in vitro stability up to 2-hour incubation at 37 °C. Small animal PET quantitation of tumor and major organs was realized by pixel analysis of regions of interest. Uptake in PC3 tumors, muscle, heart, liver, and kidney at 60 min. post injection was calculated to be 1.97 ± 0.50, 0.90 ± 0.20, 1.12 ± 0.20, 1.22 ± 0.29, and 7.06 ± 1.17%ID/g, respectively. When coinjected with 5 µg/g body weight of unlabeled BC71 peptide, the tracer uptake in the PC3 tumor dropped from 3.87 ± 1.05 %ID/g to 1.44 ± 0.38 %ID/g at 30 min p.i. and from 1.97 ± 0.50 %ID/g to 0.74 ± 0.15 at 60 min p.i., which demonstrate GRP78 receptor specific binding for ¹⁸F-AlF-NOTA-BC71. In the biodistribution study, the tumor/muscle ratios of ¹⁸F-AlF-NOTA-BC71 at 60 min p.i. was 4.81 ± 0.43, which is consistent with the high tumor to non-tumor contrast observed in the PET images. The bone uptake of ¹⁸F-AlF-NOTA-BC71 was only 0.27 ± 0.03 %ID/g at 2 h p.i., which proves the good in vivo stability of ¹⁸F-AlF-NOTA-BC71 against the ¹⁸F-deflourination. Taken together, ¹⁸F-AlF-NOTA-BC71 showed good stability, high tumor uptake, fast clearance from the body, and good tumor to normal organ uptake ratios.

Conclusions: NOTA-BC71 bioconjugate has been successfully prepared and labeled with Al¹⁸F in one single step of radiosynthesis. The favorable in vivo performance and the short radiosynthetic route of ¹⁸F-AlF-NOTA-BC71 warrant further optimization of the probe and the radiofluorination strategy to accelerate the clinical translation of ¹⁸F-labeled BC71 peptide for GRP78 receptor quantification for prostate cancer.

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