Site-Specific Labeling of PD-L1 Fab through Disulfide Rebridging for ImmunoPET Imaging

Immuno-positron emission tomography is a powerful tool for noninvasive in vivo visualization of antibody distribution and binding in tissues or organs. However, the current chemical toolbox for generating immunoPET probes compromises key attributes such as homogeneity, structural integrity, and batch-to-batch reproducibility. Quinoxaline bis-nitriles have been reported to site-selectively label antibodies via disulfide bond rebridging with robust efficiency and stability. In this study, we applied this approach to establish this chemistry for rapidly generating structurally defined Fab-based probes for in vivo imaging. Initially, we employed a NODAGA-functionalized bis-nitrile derivative to generate a PD-L1 PET probe for in vivo PET imaging with ⁶⁴Cu. Next, a functional handle was installed on the quinoxaline bis-nitrile scaffold to enable site-specific chemical PEGylation of Fab. This modification resulted in an immunoPET probe with reduced renal uptake and an improved signal-to-noise ratio. Overall, the results demonstrate that this labeling chemistry is robust and versatile for generating functional Fab conjugates for PET imaging in cancer research.