Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography.

Background: Overexpressed in various solid tumors, gastrin-releasing peptide receptor (GRPR) is a promising cancer imaging marker and therapeutic target. Although antagonists are preferable for the development of GRPR-targeted radiopharmaceuticals due to potentially fewer side effects, internalization of agonists may lead to longer tumor retention and better treatment efficacy. In this study, we systematically investigated unnatural amino acid substitutions to improve in vivo stability and tumor uptake of a previously reported GRPR-targeted agonist tracer, [Ga]Ga-TacBOMB2 (Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Thz-NH).

Results: Unnatural amino acid substitutions were conducted for Gln, Trp, Ala, Val, Gly and His, either alone or in combination. Out of 25 unnatural amino acid substitutions, tert-Leu (Tle) and NMe-His substitutions were identified to be preferable modifications especially in combination. Compared with the previously reported [Ga]Ga-TacBOMB2, the Tle and NMe-His derived [Ga]Ga-LW01110 showed retained agonist characteristics and improved GRPR binding affinity (K = 7.62 vs 1.39 nM), in vivo stability (12.7 vs 89.0% intact tracer in mouse plasma at 15 min post-injection) and tumor uptake (5.95 vs 16.6 %ID/g at 1 h post-injection).

Conclusions: Unnatural amino acid substitution is an effective strategy to improve in vivo stability and tumor uptake of peptide-based radiopharmaceuticals. With excellent tumor uptake and tumor-to-background contrast, [Ga]Ga-LW01110 is promising for detecting GRPR-expressing cancer lesions with PET. Since agonists can lead to internalization upon binding to receptors and foreseeable long tumor retention, our optimized GRPR-targeted sequence, [Tle,NMe-His,Thz]Bombesin(7-14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents.