Benedikt Klasen1, Euy Sung Moon2, Frank Rösch3. 1. Department of Chemistry - TRIGA site, Johannes Gutenberg University, Mainz, Germany. Electronic address: b.klasen@uni-mainz.de. 2. Department of Chemistry - TRIGA site, Johannes Gutenberg University, Mainz, Germany. Electronic address: emoon01@uni-mainz.de. 3. Department of Chemistry - TRIGA site, Johannes Gutenberg University, Mainz, Germany. Electronic address: frank.roesch@uni-mainz.de.
Abstract
BACKGROUND: Combining the advantages of both cyclic and acyclic chelator systems, AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) is well suited for complexation of various diagnostic and therapeutic radiometals such as gallium-68, scandium-44 and lutetium-177 under mild conditions. Due to its specificity for primary amines and pH dependent binding properties, squaric acid (SA) represents an excellent tool for selective coupling of the appropriate chelator to different target vectors. Therefore, the aim of this study was to evaluate radiolabeling properties of the novel bifunctional AAZTA5-SA being coupled to a model antibody (bevacizumab) in comparison to DOTA-SA, DTPA-p-Bn-SA and CHX-A″-DTPA-p-Bn-SA using the therapeutic nuclide lutetium-177. METHODS AND RESULTS: As proof-of-concept, bevacizumab was first functionalized with AAZTA5-SA, DOTA-SA, DTPA-p-Bn-SA or CHX-A″-DTPA-p-Bn-SA. After purification via fractionated size exclusion chromatography (SEC), the corresponding immunoconjugates were subsequently radiolabeled with lutetium-177 at pH 7 and room temperature (RT) as well as 37 °C. After 90 min, labeling of AAZTA5-SA-mAb resulted in almost quantitative radiochemical yields (RCY) of >98% and >99%, respectively. Formation of [177Lu]Lu-DTPA-p-Bn-SA-mAb indicated rapid labeling kinetics reaching similar yields at RT already after 30 min. Fast but incomplete radiolabeling of the CHX-A″-analogue could be observed with a yield of 74% after 10 min and no further significant increase. In contrast, 177Lu-labeling of DOTA-SA-mAb showed negligible radiochemical yields of <2% both at room temperature and 37 °C. In vitro complex stability measurements of [177Lu]Lu-AAZTA5-SA-mAb at 37 °C indicated >94% protein bound activity in human serum and >92% in phosphate buffered saline (PBS), respectively within 15 days. [177Lu]Lu-DTPA-p-Bn-SA-mAb and [177Lu]Lu-CHX-A″-DTPA-p-Bn-SA-mAb revealed similar to even slightly higher in vitro stability in both media. CONCLUSION: Coupling of AAZTA5-SA to the monoclonal antibody bevacizumab allowed for 177Lu-labeling with almost quantitative radiochemical yields both at room temperature and 37 °C. Within 15 days, the resulting radioconjugate indicated very high in vitro complex stability both in human serum and PBS. Therefore, AAZTA5-SA is a promising tool for 177Lu-labeling of sensitive biomolecules such as antibodies for theranostic applications.
BACKGROUND: Combining the advantages of both cyclic and acyclic chelator systems, AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) is well suited for complexation of various diagnostic and therapeutic radiometals such as gallium-68, scandium-44 and lutetium-177 under mild conditions. Due to its specificity for primary amines and pH dependent binding properties, squaric acid (SA) represents an excellent tool for selective coupling of the appropriate chelator to different target vectors. Therefore, the aim of this study was to evaluate radiolabeling properties of the novel bifunctional AAZTA5-SA being coupled to a model antibody (bevacizumab) in comparison to DOTA-SA, DTPA-p-Bn-SA and CHX-A″-DTPA-p-Bn-SA using the therapeutic nuclide lutetium-177. METHODS AND RESULTS: As proof-of-concept, bevacizumab was first functionalized with AAZTA5-SA, DOTA-SA, DTPA-p-Bn-SA or CHX-A″-DTPA-p-Bn-SA. After purification via fractionated size exclusion chromatography (SEC), the corresponding immunoconjugates were subsequently radiolabeled with lutetium-177 at pH 7 and room temperature (RT) as well as 37 °C. After 90 min, labeling of AAZTA5-SA-mAb resulted in almost quantitative radiochemical yields (RCY) of >98% and >99%, respectively. Formation of [177Lu]Lu-DTPA-p-Bn-SA-mAb indicated rapid labeling kinetics reaching similar yields at RT already after 30 min. Fast but incomplete radiolabeling of the CHX-A″-analogue could be observed with a yield of 74% after 10 min and no further significant increase. In contrast, 177Lu-labeling of DOTA-SA-mAb showed negligible radiochemical yields of <2% both at room temperature and 37 °C. In vitro complex stability measurements of [177Lu]Lu-AAZTA5-SA-mAb at 37 °C indicated >94% protein bound activity in human serum and >92% in phosphate buffered saline (PBS), respectively within 15 days. [177Lu]Lu-DTPA-p-Bn-SA-mAb and [177Lu]Lu-CHX-A″-DTPA-p-Bn-SA-mAb revealed similar to even slightly higher in vitro stability in both media. CONCLUSION: Coupling of AAZTA5-SA to the monoclonal antibody bevacizumab allowed for 177Lu-labeling with almost quantitative radiochemical yields both at room temperature and 37 °C. Within 15 days, the resulting radioconjugate indicated very high in vitro complex stability both in human serum and PBS. Therefore, AAZTA5-SA is a promising tool for 177Lu-labeling of sensitive biomolecules such as antibodies for theranostic applications.