To develop and characterise an affibody targeting EBV LMP1 and  to investigate its targeted therapeutic effects in Systemic Lupus Erythematosus (SLE) by elucidating the molecular mechanisms by which LMP1 engagement modulates downstream signalling and cellular proliferation.

A phage display-derived affibody library was screened against EBV LMP1 to identify binders with high affinity and specificity. Lead candidates were validated for selective binding to endogenous LMP1 at both molecular and cellular levels. EBV-positive cell models with high LMP1 expression, including lymphoblastoid cell lines and nasopharyngeal carcinoma cells, were analysed alongside EBV-negative or LMP1-deficient controls. Cells were treated with the LMP1-binding affibody or a non-binding control affibody. Cellular proliferation and cell-cycle distribution were assessed using metabolic assays, DNA synthesis analysis and flow cytometry. Transcriptomic profiling was performed to compare global gene expression between affibody-bound and control cells, and key signalling pathways and regulatory proteins were further evaluated by western blotting.

The LMP1-specific affibody selectively bound endogenous LMP1 and elicited biological effects exclusively in EBV-positive, LMP1-expressing cells, whereas EBV-negative or LMP1-deficient controls showed no comparable response, confirming functional target specificity. Transcriptomic profiling revealed distinct transcriptional reprogramming following LMP1 engagement, characterised by upregulation of EBV latent regulatory programmes linked to cell-cycle control and RNA biology. In contrast to canonical growth signalling, no activation of PI3K/AKT, Cyclin D family or NF-κB pathways was observed. Instead, affibody treatment was associated with marked suppression of SOCS1 expression and reduced JNK phosphorylation, indicating relief of inhibitory signalling constraints. These molecular alterations correlated with enhanced proliferation and prolonged S-phase progression in EBV-positive cells.

Domain-specific engagement of EBV LMP1 by an affibody promotes proliferation in EBV-positive cells through transcriptional reprogramming and relief of inhibitory signalling constraints, rather than activation of canonical growth pathways. These findings suggest that EBV LMP1-driven modulation of B-cell permissive states represents a previously underappreciated mechanism contributing to EBV-associated B-cell dysregulation relevant to SLE.