A Modular Antibody-Based Platform for PD-L1 Pretargeted SPECT Imaging and In Vitro 177Lu Cytotoxicity in a Murine Melanoma Model

Accurate non-invasive assessment of PD-L1 expression and function remains challenging due to tumor heterogeneity and the limitations of conventional biopsy-based studies. Here, we present a modular platform based on atezolizumab for PD-L1 imaging and radiopharmaceutical evaluation in melanoma models. Atezolizumab was functionalized with trans-cyclooctene to allow rapid and selective conjugation, via inverse-demand Diels–Alder, with 99mTc-HYNIC-tetrazine probes, generating in situ a SPECT probe for in vivo studies. In a syngeneic C57BL/6J model with Tm1 cells, pretargeting allowed tumor detection at 4 h with heterogeneous intratumoral uptake; a directly labeled antibody evaluated at the same time point is reported as a reference. Cy5-tetrazine-enabled fluorescence labeling was used for in vitro studies, i.e., flow cytometry and quantitative analysis of PD-L1 status under basal and interferon-γ-stimulated conditions. To analyze PD-L1 as a target for radiotherapy, a conjugate of atezolizumab with lutetium-177, via a DOTA chelator, was developed and evaluated in vitro: at 0.55 MBq, it reduced Tm1 cell viability more than free 177LuCl3, with a greater effect after interferon-γ stimulation, and the effect was further increased in combination with a PARP inhibitor. Using the same antibody for imaging and radionuclide-based cytotoxicity suggests a potential theranostic application, to be validated in vivo. This work describes a modular platform for PD-L1 interrogation that supports further radiopharmaceutical development.