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

Purpose 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. Method The designed Dar was synthesized and characterized by X-ray crystallography, 1 H/ 13 C NMR, and mass spectrometry. Dar-PSMA-617 was conjugated and radiolabeled with 68 Ga, 177 Lu, and 89 Zr. The in vivo behavior of 68 Ga/ 89 Zr-labeled Dar-PSMA-617 were evaluated using micro-PET imaging and biodistribution from image quantitation and tissue radioactivity counting, with 68 Ga/ 89 Zr-labeled NOTA/DOTA/DFO-PSMA-617 analogs as controls, respectively. The [ 177 Lu]-Dar-PSMA-617, with [ 177 Lu]-DOTA-PSMA-617 as control, was evaluated in competitive cell uptake, tumor cell internalization, and efflux studies. The treatment efficacy of [ 177 Lu]Lu-Dar-PSMA-617, with [ 177 Lu]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 [ 89 Zr]Zr-Dar-KN035 nanobody, with [ 89 Zr]Zr-DFO-KN035 as control, was evaluated by micro-PET imaging and biodistribution in a mouse model bearing MC38&MC38-hPD-L1 colon cancer. Results 68 Ga, 89 Zr, and 177 Lu-radiolabeled Dar-PSMA-617 complexes were able to be produced under mild condition with high radiochemical yield and purity successfully. [ 177 Lu]Lu-Dar-PSMA-617 had higher cellular uptake yet similar internalization and efflux properties in LNCaP cells, as compared to [ 177 Lu]Lu-DOTA-PSMA-617. Micro-PET images demonstrated significantly higher tumor uptake of [ 68 Ga]Ga-Dar-PSMA-617, than that of the analog [ 68 Ga]Ga-DOTA-PSMA-617. The tumor uptake values of [ 68 Ga]Ga-Dar-PSMA-617 at multiple time points are comparable to that of [ 68 Ga]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 89 Zr, while the resultant [ 89 Zr]Zr-Dar-PSMA-617 demonstrated a similar biodistribution with [ 89 Zr]Zr-DFO-PSMA-617 measured at 96 h p.i. The treatment with [ 177 Lu]Lu-Dar-PSMA-617 significantly inhibited the tumor growth, showing much better efficacy than that of [ 177 Lu]Lu-DOTA-PSMA-617 at the same injected radioactivity and mass dose. Dar was covalently linked to KN035 nanobody and enabled radiolabeling with 89 Zr in high yield and radiochemical purity at room temperature. The resultant [ 89 Zr]Zr-Dar-KN035, with [ 89 Zr]Zr-DFO-KN035 as control, demonstrated superior tumor uptake and detection capability in PET imaging studies. Conclusion 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 68 Ga, 177 Lu, and 89 Zr 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.