Validation of intrabodies against the E6 and E7 oncoproteins: a targeted therapeutic approach for the treatment of Human Papillomavirus 16 - associated lesions

Human papillomaviruses (HPVs) are a heterogeneous group of DNA viruses, a subset of which is classified as high-risk due to their causal association with cervical cancer and other anogenital and oropharyngeal malignancies. Among these, HPV16 represents the most prevalent high-risk genotype and is responsible for the majority of HPV-associated cervical cancers. Prophylactic vaccination has proven highly effective in preventing HPV infection and is currently available against up to seven high-risk genotypes (HPV16, 18, 31, 33, 45, 52, 58) and the low-risk types 6 and 11. However, these vaccines have no therapeutic effect on established infections or HPV-driven lesions and cancers. In addition, incomplete vaccine coverage and the continued circulation of oncogenic HPV genotypes not included in current vaccines highlight a significant unmet clinical need, underscoring the urgency of developing targeted therapeutic strategies for HPV-associated disease. Among viral determinants of high-risk HPVs, E6 and E7 oncoproteins are constitutively expressed in HPV-infected cells and promote malignant transformation by inactivating p53 and pRb tumor suppressors, respectively, thus impairing multiple host cellular processes. Recombinant antibodies in single-chain format expressed intracellularly – intrabodies - can represent a promising strategy to selectively neutralize viral oncoproteins and restore tumor suppressor functions. The work reported in this thesis examines the molecular and functional impact of two intrabodies developed at Istituto Superiore di Sanità against the oncoproteins of the high risk HPV16, on transfected HPV16-positive SiHa cells. When expressed intracellularly, the anti-16E6 I7NUC intrabody and the anti-16E7 43M2SD intrabody are localized in nucleus and ER, respectively. Through comprehensive transcriptomic profiling (RNA-seq) of intrabody-expressing SiHa cells, we observed sharp differences between the effects of the two intrabodies. I7NUC induced an extensive and coordinated reprogramming of gene expression, with more than twelve thousand differentially expressed genes. Pathways linked to proteostasis, nucleocytoplasmic trafficking, ER protein processing, chromatin remodelling, senescence, ubiquitin-mediated proteolysis, ribosome biogenesis, and metabolic activity were downregulated. Network analysis revealed that numerous downregulated genes encode direct interactors of E6, suggesting that I7NUC could effectively disrupt the E6-centered oncogenic interaction network. Conversely, 43M2SD elicited a markedly narrower transcriptional footprint, modulating approximately one thousand genes and significantly enriching only a single pathway, related to cytokine–receptor interactions. No direct E7 interactors were detected among its differentially expressed genes, indicating a more limited or indirect influence on cellular signaling. These findings underscore the distinct biological consequences of targeting E6 in the nucleus versus sequestering E7 in the ER compartment. To overcome the limitations of two-dimensional cell cultures, we extended our analyses to three-dimensional (3D) SiHa spheroids. They recapitulate key structural and physiological features of solid tumors, such as oxygen and nutrient gradients, hypoxic cores, and spatial heterogeneity. In this model, I7NUC displayed a rapid and pronounced expression peak corresponding to inhibition of spheroid growth. RT-qPCR analyses at different time points confirmed the activation of p53 pathway and revealed a dynamic IFN-γ-mediated response, characterized by early upregulation of IFN-γ and downstream effectors followed by late attenuation. Additionally, prolonged I7NUC expression resulted in downregulation of genes related to ER stress signalling and ferroptotic defence, suggesting a progressive weakening of adaptive survival pathways within the tumor-like environment. In contrast, 43M2SD showed slower accumulation, higher variability, and modest effects on spheroid volume, suggesting low efficacy and the need to optimize delivery. Overall, this work demonstrates that nuclear inhibition of E6 by I7NUC triggers a multifaceted antitumor program involving restoration of p53 function, suppression of oncogenic pathways, interference with metabolic and proteostatic circuits, and modulation of intrinsic immune signalling. The activity of I7NUC in 3D spheroids further supports its potential as a therapeutic candidate for HPV16-associated malignancies. More broadly, the study highlights the relevance of intrabody-based strategies for targeting viral oncogenes and underscores the utility of 3D spheroid models as physiologically relevant platforms for evaluating targeted antiviral and anticancer interventions.