I-Labeled Dual Immune Checkpoint Inhibitors for Tumor Microenvironment Improvement To Enhance Antitumor Efficacy.

Bispecific immune checkpoint inhibitors (ICIs), with their ability to target multiple immune checkpoint molecules, demonstrate promising potential as carriers for radioimmunotherapy (RIT). We used I-labeled cadonilimab (AK104), a PD-1/CTLA-4 tetravalent bispecific antibody, as a novel radio-immunotherapeutic agent for the treatment of non-small-cell lung cancer (NSCLC) and explored its underlying therapeutic mechanism. SPECT/CT imaging was performed to evaluate the systemic and tumor distributions of [I]I-AK104. The therapeutic effect of [I]I-AK104 in tumor-bearing mice was evaluated by tumor growth monitoring and histopathology analysis. Imaging mass cytometry and single-cell RNA sequencing were used to analyze the changes in the tumor microenvironment after the [I]I-AK104 treatment. [I]I-AK104 exhibited good tumor-targeting retention ability and antitumor effect. It promoted CD8 T cell infiltration and antigen-specific clonal expansion in the tumor microenvironment (TME). Furthermore, it induces immunogenic cell death (ICD) by recruiting neutrophils and triggering the release of reactive oxygen species (ROS) and nitric oxide (NO). Additionally, [I]I-AK104 enhanced interactions among immune cell subsets, increasing the prevalence of the cell neighborhood (CN) composed of neutrophils, CD8 T cells, and dendritic cells (DCs). The combination of I-based radiotherapy and bispecific ICIs remodels the TME and enhances the therapeutic efficacy in NSCLC. CD8 T cell activation and neutrophil-mediated ROS and NO release are key mechanisms contributing to enhanced tumor cell killing.