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Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) latent infection and is common in Southern China and Southeast Asia. The viral latent membrane proteins LMP1 and LMP2 are persistently expressed in NPC tissues; the cytoplasmic domain of LMP1 (LMP1 C-terminal) and LMP2A (LMP2A N-terminal) proteins is essential for maintenance of latency and can alter host cell signaling to facilitate tumor growth and progression. Thus, targeting LMP1 or LMP2 oncoprotein has been an increasing interest for diagnosis and targeted therapy of NPC. Affibody molecules, a new class of small-affinity engineered scaffold proteins, have demonstrated high potential for therapeutics, diagnostics, and biotechnological applications. More recently, radiolabelled HER2-specific affibody molecules have demonstrated to be useful in imaging of HER2 expressing tumor. In this study, we report three novel EBV LMP1 C-terminal (EBV LMP1-C) domain affibody molecules (Z LMP1-C 15, Z LMP1-C 114, and Z LMP1-C 277) were selected by biopanning from a random-peptide displayed phage library and used for molecular imaging in tumor-bearing nude mice. Surface plasmon resonance (SPR), indirect immunofluorescence, and immunohistochemistry (IHC) clearly showed that all three selected affibody molecules have high affinity and specificity in binding to EBV LMP1 protein. Moreover, in vivo tumor imaging revealed that Dylight-755-labeled affibody molecules accumulated rapidly in tumor site after injection (1 h) and then were continuously maintained for 24 h in EBV-positive NPC xenograft mice model. In conclusion, our findings highlight the potential use of Z LMP1-C affibody molecules as tumor-specific molecular imaging agents of EBV-associated NPC.Key points• We screened three novel affibody molecules (Z LMP1-C 15, Z LMP1-C 114, and Z LMP1-C 277) targeting EBV LMP1-C terminal domain• Z LMP1-C recognize the recombinant and native LMP1-C with high affinity and specificity• Z LMP1-C can be used for molecular imaging., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High-risk papillomavirus (HPV) is one of the major reasons for cervical cancer, causing most lethal gynecologic malignancies worldwide. For cervical cancer progression, oncogene E7 plays vital roles and is used as one of the major targets for cervical tumor diagnosis and treatment. In the clinic, successful treatment of cervical cancer relies on diagnosing the disease at an early stage, where a late-stage diagnosis usually led to treatment failure. In this work, we designed and purified an HPV18 E7 oncogene targeting affibody, named as Z HPV18E7 , for in vitro and in vivo imaging and targeted treatment of cervical cancer. In vitro, Z HPV18E7 showed a specific targeting effect against an HPV18 positive cell line; as a contrast, the affibody did not target the HPV18 negative cell line. In vivo, we tested the bio-distribution of the affibody in mice bearing cervical cancer. The whole animal imaging analysis indicated the affibody-targeted tumor tissue specifically with 10 min after injection, and the affibody reached the highest level at tumor tissues 45 min after injection. At the 24th hour after injection, the affibody still maintained a certain level in tumor tissues compared to other organs. To test the therapeutic effect of this affibody, we modified the affibody (i.e., Z HPV18E7 ) with a clinically used anti-cancer agent (i.e., Pseudomonas exotoxin). In a mice cervical cancer model, Z HPV18E7 was able to deliver Pseudomonas exotoxin to tumor tissues effectively, showing great potential for cancer treatment. This study indicated that Z HPV18E7 could be employed for in vitro imaging and targeted treatment of cervical cancer. Beyond the chemotherapeutic agent used in this work, the affibody could be extended for carrying other therapeutic agents for cervical cancer treatment.