AI driven approaches in Nanobody Epitope Prediction: Are We There Yet?

ABSTRACT Nanobodies have emerged as a versatile class of biologics with promising therapeutic applications, driving the need for robust tools to predict their epitopes, a critical step for in silico affinity maturation and epitope-targeted design. While molecular docking has long been employed for epitope identification, it requires substantial expertise. With the advent of AI driven tools, epitope identification has become more accessible to a broader community increasing the risk of models’ misinterpretation. In this study, we critically evaluate the nanobody epitope prediction performance of two leading models: AlphaFold3 and AlphaFold2-Multimer (v.2.3.2), highlighting their strengths and limitations. Our analysis revealed that the overall success rate remains below 50% for both tools, with AlphaFold3 achieving a modest overall improvement. Interestingly, a significant improvement in AlphaFold3’s performance was observed within a specific nanobody class. To address this discrepancy, we explored factors influencing epitope identification, demonstrating that accuracy heavily depends on CDR3 characteristics, such as its 3D spatial conformation and length, which drive binding interactions with the antigen. Additionally, we assessed the robustness of AlphaFold3’s confidence metrics, highlighting their potential for broader applications. Finally, we evaluated different strategies aimed at improving prediction success rate. This study can be extended to assess the accuracy of emerging deep learning models adopting a similar approach to AlphaFold3.