In Vitro and In Vivo Comparison of Random versus Site-Specific\nConjugation of Bifunctional Chelating Agents to the CD33-Binding Antibody\nfor Use in Alpha- and Beta-Radioimmunotherapy

Radiometal chelator\nconjugation is a cornerstone of radioimmunotherapy\n(RIT). Continued interest in selective placement of chelators remains\nan active topic of discussion in the field. With several simple site-specific\nmethods being recently reported, it was of interest to investigate\nthe benefits and potential drawbacks of the site-specific method with\na full comparison to a more typical random conjugation method that\nis currently utilized in clinical applications. In this study, the\nconjugation methods were evaluated side by side to determine the utility\nof both methods using commercially available random and site-specific\nconjugation reagents by performing antigen binding; radiolabeling\nwith <sup>64</sup>Cu, <sup>177</sup>Lu, and <sup>225</sup>Ac radioisotopes;\nantibody-conjugate stability, cytotoxicity, in vivo distribution,\npharmacokinetics analyses, and dosimetry to gather a whole data set\nfor preclinical investigation. Evaluation revealed that both methods\nperformed similarly during most experiments with the site-specific\nmethod, resulting in higher binding capacity of the antibody conjugate\nvia flow cytometry. Radiolabeling was not significantly different\nbetween two methods, while stability showed that the site-specifically\nconjugated antibody was somewhat more stable at 37 °C in human\nserum over 1 week. In vitro experiments demonstrated less cell killing\nwith the random conjugation method, while in vivo experiments showed\nno statistical differences in tumor uptake between conjugation methods.\nDosimetry calculations were performed using the acquired PET/CT data\nand showed that apart from the liver, there was no significant difference\nin radiation doses delivered by either antibody conjugate. These results\ndemonstrate that both methods are viable for future work, while the\nsite-specific method offers several potential advantages and, in some\ncases, improved efficacy.