Genetically Encoded In Vivo Ligation‐Driven Targeted Drug Delivery System for Oncotheranostics

Nanotechnology is transforming cancer diagnostics and treatment through targeted drug delivery. However, the clinical use of nanoparticles faces major challenges, including inconsistent chemical synthesis, low effectiveness, low delivery efficiency, and unintended harm to healthy cells. A promising approach to ensuring synthesis reproducibility is genetically encoded, biocompatible, self-assembling protein nanoparticles, including naturally occurring ones, while poor tumor accumulation can be mitigated through time-gated intravenous two-step injection. Here, we show the development of a first-in-class, fully genetically encoded two-step drug delivery system (DDS) for cancer treatment. The DDS realizes in vivo ligation through tumor pre-targeting with a non-toxic affibody fusion protein and utilizes Thermotoga maritima encapsulin nanoparticles to target HER2-overexpressing tumors, making it the first encapsulin-based nanoplatform for in vivo therapy. In this system, the affibody fusion protein first accumulates at the tumor site, followed by the delivery of doxorubicin-loaded encapsulin, which binds to the affibody via a SpyTag/SpyCatcher ligation system. The resulting proteinaceous nanosystem demonstrated high specificity, exhibiting an order of magnitude increase in binding to HER2+ cells compared to control cells. In vivo studies confirmed the efficacy of this two-step DDS, successfully inhibiting tumor growth by 95%, compared to 62.3% for one-step delivery and 87.5% for free doxorubicin (Dox). Moreover, it completely eliminated heart toxicity, a common side effect of free Dox, while maintaining strong anti-cancer effects. In summary, our study introduces a novel targeted drug delivery approach that addresses key challenges in nanoparticle-based treatments, such as poor tumor accumulation and inconsistent reproducibility, paving the way for more effective and safer cancer therapies.