Harnessing Fc-Directed Bioconjugation for the Synthesis of Site-Specifically Modified Radioimmunoconjugates

A growing body of preclinical and clinical evidence has shown that site-specifically and site-selectively modified immunoconjugates exhibit improved in vivo performance compared to their stochastically modified cousins. However, extant approaches to site-specific bioconjugation suffer from a variety of issues that make clinical translation challenging, including instability, complexity, and expense. Herein, we describe a novel chemical approach to the synthesis of site-specifically modified radioimmunoconjugates that is simple and straightforward. To this end, we leveraged an Fc-directed peptide to append free sulfhydryl moieties to unique sites within the Fc domain of the CA19-9-targeting antibody 5B1. These thiols were then modified with phenyloxadiazolyl methylsulfone-bearing variants of the chelator CHX-A″-DTPA, and the immunoconjugate was radiolabeled with [¹⁷⁷Lu]Lu³⁺ to produce [¹⁷⁷Lu]Lu-DTPA-^PODSAJICAP-5B1 in high yield, purity, and specific activity. Subsequent analyses confirmed the site-specificity of the modification and demonstrated the high stability and immunoreactivity of the radioimmunoconjugate. Biodistribution studies in athymic nude mice bearing subcutaneous BxPC3 pancreatic cancer xenografts revealed that [¹⁷⁷Lu]Lu-DTPA-^PODSAJICAP-5B1 produced high activity concentrations in tumor tissue as well as high tumor-to-background activity concentration ratios and displayed performance that compared favorably to ¹⁷⁷Lu-labeled analogues synthesized with traditional stochastic and thiol-selective bioconjugation strategies.