Thermosensitive and tumor-targeted asparaginase fusion protein enables metabolic-immunotherapy for solid tumors

Metabolic therapies targeting tumor amino acid dependence, represented by asparaginase (ASNase), have shown limited efficacy in solid tumors, owing to poor stability, low penetration, and high immunogenicity. Here, we design a thermosensitive tumor-targeted ASNase fusion protein (ASNase-ELP-LHRH) via AlphaFold2, by integrating elastin-like polypeptide (ELP) and luteinizing hormone-releasing hormone (LHRH), to overcome these drawbacks. ELP drives in situ depot formation, extending half-life to ∼400 h (95-fold increase) and reducing anti-ASNase antibody levels 70-fold. LHRH mediates receptor-dependent tumor targeting and deep penetration. ASNase-ELP-LHRH achieves persistent asparagine (Asn) depletion, counteracting metabolic adaptation to induce nutrient starvation and apoptosis. Mechanistically, the construct blocks metastasis by suppressing epithelial-mesenchymal transition through downregulating Twist1 and upregulating E-cadherin, effectively inhibiting lung metastasis for 25 days in triple-negative breast cancer models. We further observed time-dependent changes in CD8⁺ T-cell effector-marker expression under Asn deprivation, characterized by dynamic alterations in IFN-γ and granzyme B expression. Combined with anti-PD-L1, this achieves a "dual boost" of antitumor immunity, durably inhibiting metastasis for 40 days and extending survival 1.5-fold. The modular ligand-exchangeable platform offers a generalizable strategy for protein optimization and establishes ASNase-ELP-LHRH as a promising metabolic-immunotherapeutic agent for solid tumors, bridging protein engineering, tumor metabolism and cancer immunology.