Design, synthesis, and preclinical evaluation of a novel bifunctional macrocyclic chelator for theranostics of cancers

This study was to design and synthesize a novel bifunctional chelator, named Dar, primarily validated by conjugating to tumor targeting motifs, labeled with radiometals, and performed preclinical evaluation of tumor imaging and cancer therapy in murine tumor models.

The designed Dar was synthesized and characterized by X-ray crystallography, ¹H/¹³C NMR, and mass spectrometry. Dar-PSMA-617 was conjugated and radiolabeled with ⁶⁸Ga, ¹⁷⁷Lu, and ⁸⁹Zr. The in vivo behavior of ⁶⁸ Ga/⁸⁹Zr-labeled Dar-PSMA-617 were evaluated using micro-PET imaging and biodistribution from image quantitation and tissue radioactivity counting, with ⁶⁸Ga/⁸⁹Zr-labeled NOTA/DOTA/DFO-PSMA-617 analogs as controls, respectively. The [¹⁷⁷Lu]-Dar-PSMA-617, with [¹⁷⁷Lu]-DOTA-PSMA-617 as control, was evaluated in competitive cell uptake, tumor cell internalization, and efflux studies. The treatment efficacy of [¹⁷⁷Lu]Lu-Dar-PSMA-617, with [¹⁷⁷Lu]Lu-DOTA-PSMA-617 as control, was evaluated in PSMA-positive LNCaP tumor-bearing mice. In addition, the ability of Dar for radiolabeling nanobody was tested by conjugating Dar to KN035 nanobody. The resultant [⁸⁹Zr]Zr-Dar-KN035 nanobody, with [⁸⁹Zr]Zr-DFO-KN035 as control, was evaluated by micro-PET imaging and biodistribution in a mouse model bearing MC38&MC38-hPD-L1 colon cancer.

⁶⁸Ga, ⁸⁹Zr, and ¹⁷⁷Lu-radiolabeled Dar-PSMA-617 complexes were able to be produced under mild condition with high radiochemical yield and purity successfully. [¹⁷⁷Lu]Lu-Dar-PSMA-617 had higher cellular uptake yet similar internalization and efflux properties in LNCaP cells, as compared to [¹⁷⁷Lu]Lu-DOTA-PSMA-617. Micro-PET images demonstrated significantly higher tumor uptake of [⁶⁸Ga]Ga-Dar-PSMA-617, than that of the analog [⁶⁸Ga]Ga-DOTA-PSMA-617. The tumor uptake values of [⁶⁸Ga]Ga-Dar-PSMA-617 at multiple time points are comparable to that of [⁶⁸Ga]Ga-NOTA-PSMA-617, although a higher and persistently prolonged kidney retention was resulted in during the study period. The Dar chelator can also successfully mediate the radiolabeling with ⁸⁹Zr, while the resultant [⁸⁹Zr]Zr-Dar-PSMA-617 demonstrated a similar biodistribution with [⁸⁹Zr]Zr-DFO-PSMA-617 measured at 96 h p.i. The treatment with [¹⁷⁷Lu]Lu-Dar-PSMA-617 significantly inhibited the tumor growth, showing much better efficacy than that of [¹⁷⁷Lu]Lu-DOTA-PSMA-617 at the same injected radioactivity and mass dose. Dar was covalently linked to KN035 nanobody and enabled radiolabeling with ⁸⁹Zr in high yield and radiochemical purity at room temperature. The resultant [⁸⁹Zr]Zr-Dar-KN035, with [⁸⁹Zr]Zr-DFO-KN035 as control, demonstrated superior tumor uptake and detection capability in PET imaging studies.

The Dar, as a novel bifunctional chelator for medicating the labeling of radiometals onto tumor targeting carriers, was successfully synthesized and chemically characterized. Test radiolabeling, on PSMA-617 and a nanobody as tool targeting molecule carriers, demonstrated the Dar has potential as a universal bifunctional chelator for radiolabeling various radiometals (at least ⁶⁸Ga, ¹⁷⁷Lu, and ⁸⁹Zr tested) commonly used for clinical imaging and therapy. Using a novel Dar chelator results in altered in vivo behavior of the carriers even though labeled with the same nuclide. This capability makes Dar an alternative to the existing choices for radiolabeling new carrier molecules with various radiometals, especially the radiometals with large radius.