Purpose <p>A theranostic nanomedicine for CD3⁺ bispecific antibodies targeting glycoprotein-100 (GP-100) was tested in vivo using two radiation sessions. CT-imageable nanoparticles composed of hyaluronate-alginate and designed to release their contents upon radiation exposure were evaluated in a mouse model of B16-melanoma model in the left hind leg with pulmonary metastases.</p> Materials and Methods <p>In session 1, IFN-γ was encapsulated during the Fe polymerization of hyaluronate-alginate nanoparticles. Nine hours after the intravenous injection of 1 × 10¹⁰ IFN-γ nanoparticles, enough to observe dose escalation of either 10 or 20&#xa0;Gy was administered using 140&#xa0;keV-X-ray to the primary and metastatic tumors. In session 2, tebentafusp was encapsulated using the same method as in session 1. Seventy-two hours after the intravenous injection of 1 × 10¹⁰ tebentafusp-loaded nanoparticles, radiation was administered under conditions identical to those in session 1.</p> Results <p>In session 1, IFN-γ-loaded nanoparticles selectively accumulated in the primary tumor and pulmonary metastasis by passing through the coarse endothelium of tumor vasculature, which could be visualized using CT. IFN-γ nanoparticles continuously released IFN-γ, facilitating the formation of the HLA-A*02:01-GP100-complex. In session 2, the tebentafusp-loaded nanoparticles continuously released tebentafusp, leading to the formation of an immunological synapse consisting of HLA-A*02:01-GP100, tebentafusp, and CD3 on T cells. CD3⁺ T cells release perforin and granzymes, resulting in the cytolysis of the primary tumor and pulmonary metastasis. This effect was synergistic with that of radiation, resulting in Enhancement Factor (EF) more than 1.</p> Conclusion <p>Theranostic nanomedicine demonstrated potential as a dual therapeutic and diagnostic strategy for targeting tumors and metastases, with synergistic effects observed when combined with radiation.</p> Graphical abstract <p></p>