Project Summary/Abstract For over three decades, the ligation between maleimide-bearing probes and thiols has been a cornerstone of the synthesis of site-specifically labeled antibodies. Yet despite its popularity, this approach to bioconjugation has serious limitations. Maleimide-based conjugates display limited stability in vivo because their succinimidyl thioether linkage can undergo a retro-Michael reaction that leads to the dissociation of the cargo or its exchange with circulating biomolecules. In the context of nuclear imaging and radioimmunotherapy, this retro- Michael reaction can lead to the release of the radioactive payload and the in vivo radiolabeling of endogeneous biomolecules, resulting in higher radiation doses to healthy tissues, reduced imaging contrast, and lower therapeutic ratios. In order to circumvent this obstacle, we have developed a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent ? `PODS' ? as a platform for thiol-based bioconjugations. We have demonstrated that PODS can be used to reproducibly create homogenous, well- defined, highly immunoreactive, and highly stable radioimmunoconjugates with far superior in vivo performance compared to analogous probes synthesized via traditional, maleimide-based approaches. This proposal is centered upon the expansion and optimization of this technology. We will seek to build upon our preliminary work by optimizing the rapidity and selectivity of the PODS-thiol ligation and evaluating the in vivo performance of PODS-based radioimmunoconjugates labeled with 89Zr, 177Lu, 131I, and 225Ac. Specific Aim 1 (SA1) will be focused on the design, synthesis, and characterization of a library of second- generation PODS reagents, with the overall goal of identifying a single construct with the most favorable combination of stability, solubility, and reactivity. Specific Aim 2 (SA2) will be centered on the evaluation of the in vivo performance of radioimmunoconjugates synthesized using PODS. The most promising second- generation PODS reagent from SA1 will be used to synthesize 89Zr- and 177Lu-labeled radioimmunoconjugates, and their in vivo performance will be assessed in mouse models of cancer and compared to that of radiolabeled antibodies created using maleimide-based bioconjugations. Specific Aim 3 (SA3) will focus on the development of PODS-based prosthetic groups for the site-specific radiolabeling of antibodies with 131I and 225Ac. The in vivo performance of 131I- and 225Ac-labeled antibodies synthesized using these prosthetic groups will be evaluated in murine models of cancer and compared to analogues synthesized using current `gold- standard' strategies. We believe that this proposal could have a significant near-term impact on clinical care by developing and validating tools for the synthesis of highly stable radioimmunoconjugates with excellent in vivo performance, thereby improving imaging protocols, treatment regimens, and patient outcomes. Furthermore, we contend that this work could have a paradigm-shifting influence on bioconjugation chemistry, fundamentally changing the way biomolecular medicines are synthesized in the laboratory and clinic.