Development of Dar3A Chelator and Construction of FAP-Targeted PET Probe as Proof of Concept and for First-in-Human Evaluation

Developing novel radiopharmaceuticals for cancer theranostics has recently attracted extensive interest. Bifunctional chelators are key components of many radiometal-based radiopharmaceuticals. For the established ⁶⁸Ga/¹⁷⁷Lu theranostic pair, the commonly used chelator DOTA suffers from some issues, such as the requirement for heating during radiolabeling and slow chelation kinetics. The limitations highlight the need to develop new bifunctional chelators. Methods: The novel macrocyclic chelator 2-[11,27-bis(carboxymethyl)-34,36-dihydroxy-7,23-dimethyl-3,11,19,27,33,35-hexaazapentacyclo[27.3.1.1⁵,⁹.1¹³,¹⁷.1²¹,²⁵]hexatriaconta-1(33),5(36),6,8,13,15,17(35),21(34),22,24,29,31-dodecaen-3-yl]acetic acid (Dar3A) was synthesized and modified with a pendant arm for bioconjugation. A comprehensive comparison with DOTA was performed, including titration experiments to assess thermodynamic stability and radiolabeling studies to evaluate the efficiency of ⁶⁸Ga and ¹⁷⁷Lu labeling. For a proof of concept, Dar3A was conjugated with a fibroblast activation protein–targeted moiety derived from fibroblast activation protein inhibitor-04 and radiolabeled with ⁶⁸Ga to prepare [⁶⁸Ga]Ga-SMIC-3101. The resulting ⁶⁸Ga-labeled radiotracer was further evaluated in cellular assays, animal models, and human subjects (3 healthy volunteers and 1 patient with esophageal cancer). Results: Dar3A exhibited superior radiolabeling efficiency with ⁶⁸Ga and ¹⁷⁷Lu compared with DOTA, and [⁶⁸Ga]Ga-SMIC-3101 was obtained with a molar activity of 28.7 MBq/nmol. In U87MG tumor–bearing mice, [⁶⁸Ga]Ga-SMIC-3101 exhibited enhanced tumor uptake (12.73 ± 1.68 and 10.64 ± 2.88 %ID/g at 2 and 4 h, respectively), significantly exceeding that of DOTA-based [⁶⁸Ga]Ga-FAPI-04 (0.66 ± 0.22 and 0.51 ± 0.10 %ID/g, respectively). Moreover, the first-in-human study demonstrated that [⁶⁸Ga]Ga-SMIC-3101 was safe, with physiologic excretion occurring primarily via the hepatobiliary system, and clearly visualized primary and metastatic esophageal cancer lesions. Conclusion: Dar3A is a promising bifunctional chelator for radiolabeling and bioconjugation, supporting theranostic radiopharmaceutical development.