Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2(+) malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (Z(EBV LMP-2)12, Z(EBV LMP-2)132, Z(EBV LMP-2)137, and Z(EBV LMP-2)142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. Z(EBV LMP-2) affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2(+) xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the Z(EBV LMP-2) 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies. Author summary Molecular imaging diagnosis and targeted therapy have been successfully used for several types of tumors, but not yet applied to diagnose or treat EBV-associated NPC. Affibody molecules are small proteins engineered to bind to a large number of target proteins with high affinity, and therefore, can be developed as potential biopharmaceutical drugs for molecular diagnosis and therapeutic applications. In the present study, we screened and characterized EBV LMP-2-specific affibodies and evaluated their usage in molecular imaging of LMP-2 expressing cells and EBV LMP-2 tumor-bearing mice. Subsequently, we engineered and obtained an EBV LMP-2 affitoxin based on EBV LMP-2-binding affibodies and demonstrated its targeted cytotoxicity for EBV+ cell lines in vitro and in vivo. Our data indicate that the EBV LMP-2-specific affibody and its derived affitoxin are useful for diagnosis of LMP-2 expressing cells and targeted therapy of EBV-derived, LMP-2(+) malignancies.

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