Abstract
Nasopharyngeal carcinoma (NPC) induced by latent infection with Epstein-Barr virus (EBV) remains the most common head and neck cancer in Southeast Asia, especially in the southern part of China. It is well known that persistent expression of two EBV latent membrane proteins (LMP1/LMP2A) plays a key role in nasopharyngeal carcinogenesis. Therefore, the therapeutic approach of targeting the LMP1/LMP2A protein and subsequently blocking the LMP1/LMP2A-mediated signalling pathway has been considered for treating patients with NPC. Recently, affibody molecules, a new class of small (~6.5 kDa) affinity proteins, have been confirmed to be powerful generalisable tools for developing imaging or therapeutic agents by targeting specific molecules. In this study, three EBV LMP2A N-terminal domain-binding affibody molecules (ZLMP2A-N85, ZLMP2A-N110 and ZLMP2A-N252) were identified by screening a phage-displayed peptide library, and their high affinity and specificity for the EBV LMP2A N-terminal domain were confirmed by surface plasmon resonance (SPR), indirect immunofluorescence, co-immunoprecipitation and near-infrared small animal fluorescence imaging in vitro and in vivo. Moreover, affibody molecules targeting the EBV LMP2A N-terminal domain significantly reduced the viability of the EBV-positive cell lines C666-1, CNE-2Z and B95-8. Further investigations showed that affibody ZLMP2A-N110 could inhibit the phosphorylation of AKT, GSK-3β and β-catenin signalling proteins, leading to suppression of β-catenin nuclear translocation and subsequent inhibition of c-Myc oncogene expression, which may be responsible for the reduced viability of NPC-derived cell lines. In conclusion, our findings provide a strong evidence that three novel EBV LMP2A N-terminal domain-binding affibody molecules have great potential for utilisation and development as agents for both molecular imaging and targeted therapy of EBV-related NPC.
Highlights
Introduction EpsteinBarr virus (EBV), known as human gammaherpesvirus (HHV-4), was discovered by Epstein andBarr in tumour cell cultures derived from endemic Burkitt lymphoma (BL) patients in 19641
SDS-PAGE analysis showed that the purity of the final products was approximately 95% (Fig. 1d), meaning that these products could be used for subsequent investigations
Given that the ZLMP2A-N affibodies were able to bind to recombinant LMP2A-NCD in the surface plasmon resonance (SPR) analysis, we investigated whether the ZLMP2A-N affibodies could bind to native LMP2A-NCD in Epstein-Barr virus (EBV)-positive cells using an indirect immunofluorescence assay (IFA)
Summary
Barr in tumour cell cultures derived from endemic Burkitt lymphoma (BL) patients in 19641 This virus is exceptionally prevalent in humans, and its specific serum antibody can be detected in more than 95% of adults worldwide[2,3]. Several human cancers, such as BL, nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma (HL). Zhu et al Cell Death and Disease (2020)11:213 high prevalence in Southeast Asia, especially in the southern part of China[7,8] Both radiotherapy and chemotherapy are commonly used in the treatment of NPC patients; the prognosis remains poor due to local recurrence and distant metastasis[9]. The development of diagnostic and treatment methods, such as molecular imaging probes and targeting agents, is urgently needed to improve the early diagnosis and clinical outcome of NPC patients
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