Divergent Functional Checkpoints Determine Epitope-Specific ADE Outcomes of SARS-CoV-2 Entry

Accumulating in vitro evidence indicates that certain antibodies promote Fcγ receptor (FcγR)-dependent entry of SARS-CoV-2 into immune cells, and that the magnitude of antibody-dependent enhancement (ADE) is strongly dictated by epitope specificity. However, the mechanistic basis underlying epitope-specific ADE outcomes remains poorly understood. Here, we systematically evaluated ADE of SARS-CoV-2 entry mediated by a panel of 40 monoclonal antibodies (mAbs) targeting major antigenic regions of the spike (S) protein. Using a stepwise functional framework, we assessed antibody performance across successive stages of the FcγR-dependent viral entry pathway, including spike binding, cell-surface attachment, internalization, and membrane fusion. Although all mAbs exhibited strong binding to the S protein, their ability to support subsequent steps varied markedly by epitope. Specifically, receptor-binding domain (RBD) class I- and S2-targeting mAbs failed to support cell-surface attachment; RBD class IV-targeting mAbs and a subset of N-terminal domain (NTD)-targeting mAbs supported attachment but not internalization; and mAbs targeting a specific sub-epitope within RBD class III supported both attachment and internalization but failed to promote membrane fusion. Consequently, antibodies stalling at any of these checkpoints displayed an ADE-negative phenotype. In contrast, antibodies targeting the identified ADE-positive epitopes (RBD class II, certain NTD, and specific RBD class III sub-epitope) successfully fulfilled the functional requirements at each stage of the entry pathway. Importantly, authentic SARS-CoV-2 experiments confirmed the epitope-specific pattern of viral entry enhancement. These findings establish that divergent functional checkpoints causally determine epitope-specific ADE outcomes, providing a mechanistic framework that directly informs the design of safer therapeutic antibodies and vaccines.