Development of a 177Lu-labeled EphA2-targeting cyclic peptide combined with an HPK1 inhibitor for synergistic anti-tumor effects.

PURPOSE: Despite advancements in 177Lu-based radiotherapy for cancer, its efficacy against refractory cold tumors remains limited. Targeted peptide-radionuclide conjugates (PRCs) combined with immunotherapy are emerging as promising theranostic strategies to maximize anti-tumor effectiveness. METHODS: Cyclic peptide CEMJ4 was identified via phage selection, and further conjugated with DOTA and radiolabeled with 68Ga for diagnostic imaging and 177Lu for cancer therapy. The druggability was assessed by in vitro cell experiments, in vivo PET/CT imaging and biodistribution. Additionally, the feasibility of combining 177Lu-DOTA-CEMJ4 with a hematopoietic progenitor kinase 1 inhibitor (HPK1i) was evaluated in B16F10 tumor-bearing mice, focusing on the anti-tumor immune response and tumor growth. RESULTS: CEMJ4 exhibited high affinity for human erythropoietin-producing hepatocellular receptor A2 (EphA2; KD = 0.3 ± 0.2 µM), a therapeutic target overexpressed in several solid tumors. Radiolabeled 68Ga/177Lu-DOTA-CEMJ4 specifically bound to EphA2-expressing B16F10 cells and tumor models, effectively inhibiting tumor growth. Notably, 177Lu-induced T cell immunotoxicity was reversed by HPK1i, which modulated T cell dysfunction. Combining 177Lu-DOTA-CEMJ4 with HPK1i significantly reduced tumor burden and increased tumor-infiltrating CD4+ T cells, CD8+ T cells, and M1 macrophages. CONCLUSION: This study identifies CEMJ4 as a promising peptide ligand for tumor-targeted radionuclide delivery and emphasizes the clinical potential of radionuclide therapy combined with immunotherapy in theranostics to enhance therapeutic precision and efficacy.