Novel Bifunctional Affibody Molecules with Specific Binding to Both EBV LMP1 and LMP2 for Targeted Therapy of Nasopharyngeal Carcinoma

Antibodies are considered highly specific therapeutic agents in cancer medicines, and numerous formats have been developed. Among them, bispecific antibodies (BsAbs) have gained a lot of attention as a next-generation strategy for cancer therapy. However, poor tumor penetration is a major challenge because of their large size and thus contributes to suboptimal responses within cancer cells. On the other hand, affibody molecules are a new class of engineered affinity proteins and have achieved several promising results with their applications in molecular imaging diagnostics and targeted tumor therapy. In this study, an alternative format for bispecific molecules was constructed and investigated, named Z(LMP)110-277 and Z(LMP)277-110, that targets Epstein-Barr virus latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2). Surface plasmon resonance (SPR), indirect immunofluorescence assay, co-immunoprecipitation, and near-infrared (NIR) imaging clearly demonstrated that Z(LMP)110-277 and Z(LMP)277-110 have good binding affinity and specificity for both LMP1 and LMP2 in vitro and in vivo. Moreover, Z(LMP)110-277 and Z(LMP)277-110, especially Z(LMP)277-110, significantly reduced the cell viability of C666-1 and CNE-2Z as compared to their monospecific counterparts. Z(LMP)110-277 and Z(LMP)277-110 could inhibit phosphorylation of proteins modulated by the MEK/ERK/p90RSK signaling pathway, ultimately leading to suppression of oncogene nuclear translocations. Furthermore, Z(LMP)110-277 and Z(LMP)277-110 showed significant antitumor efficacy in nasopharyngeal carcinoma-bearing nude mice. Overall, our results demonstrated that Z(LMP)110-277 and Z(LMP)277-110, especially Z(LMP)277-110, are promising novel prognostic indicators for molecular imaging and targeted tumor therapy of EBV-associated nasopharyngeal carcinoma.