Human papillomavirus 18 E6-specific Affibody inhibits cervical cancer cell proliferation and induces apoptosis

Human papillomavirus (HPV) 18 E6 oncoprotein drives cervical carcinogenesis by degrading p53, enabling uncontrolled cell proliferation. In this study, we developed novel trans-activator of transcription (TAT)-conjugated Affibody molecules (TAT-Z_HPV18E6) targeting HPV18 E6 for therapeutic applications. High-affinity Affibody variants were screened by phage display using recombinant HPV18 E6 expressed in Escherichia coli. Three candidates (TAT-Z_HPV18E6: 4, 59, 352) exhibited high binding affinity, with equilibrium dissociation constant (K_D) ranging from 10^-6 to 10^-4 M, with TAT-Z_HPV18E6: 59 showing superior specificity for native HPV18 E6 in HeLa229 cells, as validated by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and immuno-fluorescence. In vivo near-infrared fluorescence imaging of DyLight 755-labeled TAT-Z_HPV18E6: 59 in tumor-bearing mice demonstrated rapid tumor accumulation (peak at 2 h) and prolonged retention (> 12 h). Mechanistically, TAT-Z_HPV18E6: 59 restored p53 stability and upregulated pro-apoptotic factors (Bax, PUMA) and cell cycle regulator p21. Notably, combined treatment with TAT-Z_HPV18E6: 59 and E7-targeting TAT-Z_HPV18E7: 228 combinatorial enhanced apoptosis and suppressed HeLa229 proliferation, as confirmed by CCK-8 and clonogenic assays. These results demonstrate the utility of TAT-Z_HPV18E6 Affibody molecules as targeted agents, highlighting combinatorial E6/E7 targeting as a potent strategy for HPV18-driven cervical cancer therapy. KEY POINTS: • TAT-Z_HPV18E6: 59 achieved high affinity for HPV18 E6, enabling precise molecular targeting. • Demonstrates dual utility for in vivo imaging and p53-dependent tumor suppression. • Dual targeting of E6/E7 yields synergistic therapeutic efficacy in cervical cancer models.