Preparation and Characterization of Site-Specifically Radiolabeled 89Zr-DFO-anti-PD-L1-mAb ImmunoPET Tracer
Huang, Feng-Yun Jimmy
Preparation and Characterization of Site-Specifically Radiolabeled 89Zr-DFO-anti-PD-L1-mAb ImmunoPET Tracer Feng-Yun J. Huanga,*, Ching-Chun Lua, Wei-Lin Lob, Shiou-Shiow Farnb, Chao-Wei Yangc aDepartment of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology: No.666, Buzih Road, Beitun District, Taichung City, 40605, Taiwan; bIsotope Division, Institute of Nuclear Energy Research: No.1000, Wenhua Road, Jiaan Village, Longtan District, Taoyuan City, 32546, Taiwan; cDepartment of Nuclear Medicine, Cheng Ching Hospital – Chung Kang Branch: No.966, Sec 4, Taiwan Blvd., Xitun Dist., Taichung City, 40764, Taiwan; *Corresponding Author Email Address: firstname.lastname@example.org Introduction Site-specifically radiolabeled immunoPET tracers have been demonstrated to provide superior imaging ability in vivo than conventional radiolabeled one (random method). In this study, preparation and characterization of site-specifically radiolabeled 89Zr-DFO-anti-PD-L1-mAb tracer will be investigated. The site-specific immunoPET tracer could detect the expression of immune checkpoint protein (ex. PD-L1/PD-1) on tumor for assessment of patient stratification before treatment and therapeutic efficacy after treatment. Description of the Work Materials & Methods: The technique of enzymatic glycan modification was utilized to prepare site-specifically radiolabeled 89Zr-immunoPET tracer. In brief, GlyCLICK® and SiteClick® kits were used to prepare azide-activated anti-PD-L1-mAb with degree of labeling of 2 and 4, respectively. Then bifunctional chelator DBCO-DFO was attached to the azide-functionalized antibodies via SPAAC click reaction to form site-specific DFO-anti-PD-L1-mAb conjugates with different chelator-to-antibody ratio (CAR) of 2 and 4. The quality control of conjugates were conducted and then radiolabeled with 89Zr in 1 M HEPES buffer, pH 7, and shaking with 300 rpm at RT for 40 min. In addition, in vitro stability of tracers was estimated in the PBS at RT after purification. Results: Both site-specific DFO-anti-PD-L1-mAb conjugates with CAR of 2 and 4 were performed as transparent, clear, without aggregation, chemical purity of 100%, pH of 7.0 – 7.5. Analysis of conjugates by LC-MS showed that CAR for GlyCLICK® and SiteClick® prepared DFO-anti-PD-L1-mAb conjugates was 2.04 and 3.62, respectively. Results from radio-TLC indicated that radiochemical purity of tracers with CAR of 2 and 4 reached 100% and 98.7%, respectively. In addition, the results from HPLC analysis revealed that radioimpurities in both tracers were less than 5%. For in vitro stability study, radiochemical purity of both tracers displayed no any decline until 7 d incubated in PBS at RT. Conclusions In this study, site-specifically radiolabeled 89Zr-DFO-anti-PD-L1-mAb tracer with CAR of 2 and 4 have been prepared and characterized. The LC-MS results demonstrate that CAR for GlyCLICK® and SiteClick® prepared DFO-anti-PD-L1-mAb conjugates was 2.04 and 3.62, respectively. Radiochemical purity of tracers with CAR of 2 and 4 were large than 98% and both of them showed excellent stability in vitro. Keywords: site-specifically radiolabeled; DFO; 89Zr; immunoPET; PD-L1/PD-1 References European Journal of Nuclear Medicine and Molecular Imaging, 2019;47(5):1302 – 1313.
site-specifically radiolabeled, DFO, 89Zr, immunoPET, PD-L1/PD-1