An Integrative Strategy Enhancing Nanobody Thermostability via CDR Grafting, In Silico Mutagenesis Screening, and Multiplex Evaluation

Nanobodies are transformative tools in biomedical research and therapy due to their structural advantages and exceptional stability. However, their intrinsic stability varies significantly, while existing stabilization strategies often face various limitations. Here, we report a computational–experimental integrative approach that combines complementarity-determining region (CDR) grafting with virtual mutagenesis for stabilization. Using A4.2m as the framework region donor and Nb20, a SARS-CoV-2 spike protein-targeting nanobody, as the CDR source, Nb20–4.2m was engineered and demonstrated a 10 °C enhancement in melting temperature ( T m ) and 55% improvement of refolding efficiency. Subsequently, through a computational pipeline, experimental validations, and a combination of mutations, the final construct was yielded with 68 °C T m and >82% refolding efficiency. A molecular dynamics simulation indicated that the stability enhancement originates from optimized intramolecular hydrogen bonding networks. With a higher efficiency than conventional methods, this approach offered a paradigm shift in engineering and established a versatile platform for nanobody optimization to fit broad applications in clinics and industry.