Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-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, [⁶⁸Ga]Ga-TacBOMB2 (⁶⁸Ga-DOTA-Pip-D-Phe⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Gly¹¹-His¹²-Leu¹³-Thz¹⁴-NH₂).

Results

Unnatural amino acid substitutions were conducted for Gln⁷, Trp⁸, Ala⁹, Val¹⁰, Gly¹¹ and His¹², either alone or in combination. Out of 25 unnatural amino acid substitutions, tert-Leu¹⁰ (Tle¹⁰) and NMe-His¹² substitutions were identified to be preferable modifications especially in combination. Compared with the previously reported [⁶⁸Ga]Ga-TacBOMB2, the Tle¹⁰ and NMe-His¹² derived [⁶⁸Ga]Ga-LW01110 showed retained agonist characteristics and improved GRPR binding affinity (K_i = 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, [⁶⁸Ga]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, [Tle¹⁰,NMe-His¹²,Thz¹⁴]Bombesin(7–14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents.