Fluorobenzylation promotes immune-activating peptides to turn cold tumors into hot tumors.

Abstract

Transforming immunologically "cold" tumors into "hot" lesions amenable to immunotherapy remains a central challenge. Here, we introduce fluorobenzylation as a sequence-dependent amplifier of peptide immunogenicity that enhances membrane interaction and stability. Among peptide panels, the fluorobenzylated lead FPP5 (but not scrambled controls) significantly increased ICD hallmarks (CRT, ATP, and HMGB1) relative to the native peptide and matched the doxorubicin benchmark in vitro. To enable delivery, FPP5 was assembled with FPRGD and FPPEG into composition-defined nanoparticles that are ∼150 nm and serum-stable, and exhibit a low CAC (∼10 μg mL^-1), supporting dilution-triggered intracellular disassembly and release of bioactive FPP5 after uptake. FPP5 showed enhanced penetration in 3D spheroids, and FPNPs further improved intratumoral transport via multivalency and integrin engagement. ¹⁸F-labeled FPNPs enabled PET tracking, confirming efficient tumor accumulation consistent with long-circulating nanocarrier behavior. In a breast cancer model, FPNPs potentiated ICD, increased calreticulin exposure and CD8⁺ T-cell infiltration, and synergized with anti-PD-L1 to elicit robust antitumor immunity. Collectively, these data delineate a coherent mechanism-sequence-dependent fluorobenzylation, nanoparticle delivery, intracellular release, ICD and immune activation-and establish a programmable platform to convert cold tumors and enhance combination immunotherapy.