Engineering bispecific HER2xVEGF designed ankyrin repeat proteins for targeted nanobiotechnology applications

Abstract

Background: Targeting both tumor cell surface receptors and soluble pro-angiogenic factors is a promising strategy to improve cancer treatment specificity and reduce therapy resistance. Human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) are two clinically validated targets implicated in tumor growth, metastasis, and angiogenesis. To address limitations of conventional large-molecule therapeutics, we developed a modular bispecific protein scaffold based on Designed Ankyrin Repeat Proteins (DARPins) for precision nanobiotechnology applications. Results: We engineered a bispecific HER2×VEGF DARPin containing a C-terminal cysteine for site-specific bioorthogonal conjugation. The construct was recombinantly expressed in Escherichia coli and purified to high monomeric purity. Subsequent azide functionalization enabled strain-promoted click conjugation with diverse payloads, including fluorescent dyes for imaging, radionuclide chelators for diagnostic and therapeutic isotope labeling, and a cleavable drug linker for cytotoxic payload delivery. All bioconjugates retained high structural integrity and dual-specific binding affinity to HER2 and VEGF, with dissociation constants in the low picomolar to nanomolar range as measured by surface plasmon resonance. Importantly, site-specific conjugation did not impair antigen recognition, highlighting the robustness of the scaffold. Conclusions: This study presents a scalable and versatile bispecific DARPin platform that enables modular, site-specific conjugation of imaging and therapeutic payloads without loss of binding function. The preserved dual-targeting capability and biochemical stability make it a promising candidate for nanobiotechnology-based diagnostics, radiotherapy, and targeted drug delivery in precision oncology.