SCN-PYTA:一种用于三价放射性金属的新型双功能螯合剂(BFC),用于开发靶向特异性的显像与治疗放射性药物

Introduction: The 18-membered macrocycle PYTA is a universal chelator that can complex trivalent radiometals (e.g., 225 Ac, 177 Lu, 111 In and 44 Sc) useful for imaging and targeted therapy (Chemical Sci 2024;15:11279-11286). The investigators at ORNL synthesized a BFC, SCN-PYTA, which contains an amine-reactive thiocyanate (SCN) group appended to the pyridine in the PYTA chelator. To establish PYTA as a platform to prepare theranostic radiopharmaceuticals, we investigated the conjugation characteristics of SCN-PYTA to monoclonal antibodies (mAb) and radiometals ( 111 In and 225 Ac) to define the chelation properties of PYTA-mAb conjugates. For direct comparison, labeling studies were performed using two other BFCs: SCN-DOTA and SCN-Macropa. Materials and Methods: Anti-PSMA mAb, rosopatamab and anti-TROP-2 mAb, sacituzumab solutions at 10 mg/mL were prepared in sodium carbonate buffer, pH 9.0±0.2. The BFC solutions (at 5-10 mg/mL) were prepared in metal-free water. Different molar ratios of BFC to mAb were incubated at 37 o C for 90 min. The chelate conjugated mAbs were purified by gel chromatography and 0.05 M HEPES buffer, pH 7.5. The chelator:antibody ratios (CAR) were determined using a modified colorimetric arsenazo assay. Chelate-antibody conjugates (0.025-2.0 mg) were incubated (at RT or 37 o C) with 0.05-0.5 mCi of 225 Ac or 111 In chloride in sodium acetate or tetramethylammonium acetate buffer, pH 6±0.3. Radiolabeled mAbs were purified using PD-10 gel permeation chromatography and saline solution containing 1% albumin, and assessed for radiochemical purity (RCP), stability, and immunoreactivity. Results: PYTA and Macropa conjugated mAbs complex 225 Ac or 111 In radionuclides very efficiently at RT within 5 min with labeling yields of >97%. The radiochemical purity (RCP) of purified 225 Ac-chelator-mAb preparations were >99%. In contrast, DOTA-conjugated mAbs do not label efficiently at RT. Higher labeling yield (80-95%), however, can be obtained at 37 o C with longer incubation of 1-2 hours. 225 Ac-PYTA-mAbs showed remarkably high in vitro stability at 2-8 o C for >10 days (with >99% RCP). Similar stability was observed when challenged with serum or excess chelate (EDTA and DTPA) concentrations. Based on in vitro cell binding studies using PSMA+ LNCaP cells and TROP-2+ MCF-7 cells, the immunoreactive fraction (IRF) of 225 Ac-PYTA-Rosopatamab and 225 Ac-PYTA-Sacituzumab at an infinite antigen concentration (Lindmo assay) was >99%. Conclusion: SCN-PYTA is an ideal BFC and a suitable platform to develop target specific imaging and therapeutic radiopharmaceuticals based on trivalent radiometals, peptides, proteins, and small molecules. Our preliminary data also suggests that radiolabeled sacituzumab has significant potential as a theranostic radiopharmaceutical for imaging and targeted therapy of TROP-2 positive cancers.