Abstract
Nanobodies (Nbs), miniature antibodies consisting solely of the variable region of heavy chains, exhibit unique properties such as small size, high stability, and strong specificity, making them highly promising for disease diagnosis and treatment. The engineering production of Nbs has evolved into a mature process, involving library construction, screening, and expression purification. Different library types, including immune, naïve, and synthetic/semi-synthetic libraries, offer diverse options for various applications, while display platforms like phage display, cell surface display, and non-surface display provide efficient screening of target Nbs. Recent advancements in artificial intelligence (AI) have opened new avenues in Nb engineering. AI's exceptional performance in protein structure prediction and molecular interaction simulation has introduced novel perspectives and tools for Nb design and optimization. Integrating AI with traditional experimental methods is anticipated to enhance the efficiency and precision of Nb development, expediting the transition from basic research to clinical applications. This review comprehensively examines the latest progress in Nb engineering, emphasizing library construction strategies, display platform technologies, and AI applications. It evaluates the strengths and weaknesses of various libraries and display platforms and explores the potential and challenges of AI in predicting Nb structure, antigen-antibody interactions, and optimizing physicochemical properties.