Targeted a-Therapy Using 225Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment

Targeted radionuclide therapy (TRT) using targeting moieties labeled with a-particle-emitting radionuclides (a-TRT) is an intensely investi-gated treatment approach as the short range of a-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of a-TRT is lacking in literature. Methods: Using flow cytometry of tumors, sple-nocyte restimulation, and multiplex analysis of blood serum, we stud-ied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with a-TRT and increased blood levels of various cytokines such as interferon-g, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on a-TRT. At the tumor site, a-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protu-moral alternatively activated macrophages and an increase in antitu-moral macrophages and dendritic cells. We also showed that a-TRT increased the percentage of programmed death-ligand 1 (PD-L1)- positive (PD-L1pos) immune cells in the TME. To circumvent this im-munosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combi-nation of a-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from a-TRT. Conclusion: These data suggest that a-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of a-TRT. However, further optimization is warranted to avoid adverse events.