Targeted degradation of the HPV oncoprotein E6 reduces tumor burden in cervical cancer

Abstract

Human papillomavirus (HPV) remains a global health burden, yet there are no targeted therapies for HPV-related cancers. The HPV E6 protein is essential for tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting proteolysis targeting chimera (PROTAC) that inhibits the growth of HPV⁺ tumors. To develop E6 antagonists, we generated a panel of nanobodies targeting HPV16 E6 protein. Nanobody A5 was fused to Von Hippel-Lindau protein to generate a PROTAC that degrades E6 (PROTAC^E6). Mutational rescue experiments validated E6 degradation via the CRL2^VHL E3 ligase. To deliver PROTAC^E6, we used a clinically viable DNA vaccine, which offers the advantages of localized PROTAC^E6 expression and low production costs compared to protein- or viral-based therapies. Intralesional administration of the PROTAC^E6 reduced tumor burden in an immunocompetent mouse model of HPV⁺ cancer. The PROTAC^E6 inhibitory effect was abrogated by CD4⁺ and CD8⁺ T cell depletion, indicating that the antitumor function of the PROTAC^E6 relies in part on host immune responses. These results demonstrate that E6 degradation inhibits its oncogenic function and stimulates an immune response in HPV⁺ tumors, opening new opportunities for virus-specific therapies in the treatment of HPV-related cancers.

Keywords: E6; HPV; antiviral; bioPROTAC; biologics; cancer; nanobody; oncoproteins.

Figure 1.. Identification of nanobodies to HPV16 E6.

(a) Schematic overview of the selection workflow used to isolate E6-binding nanobodies from a synthetic yeast surface display library. (b) Selection strategy included sequential rounds of magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), colony screening, and sequencing. (c) Flow cytometry histogram plots showing progressive enrichment of E6-binding yeast clones across selection rounds. Yeast were stained with fluorescently labeled E6 protein (50nM) following each round (Rd) of selection. (d) Dot plots of 11 unique nanobody clones that bind to E6.

Figure 2.. Biophysical characterization of nanobodies to HPV16 E6.

(a) Melting temperatures for all nanobodies, analyzed using DSF (N=3, mean ± SD). (b) Detection of E6-nanobody complex by MBP-pulldown. MBP-tagged nanobodies were co-expressed with His-tagged HPV16E6. Nanobody complexes were immobilized on amylose resin and resolved by Coomassie-stained SDS-PAGE gel. (c) Microscale thermophoresis (MST) binding curves for nanobodies interacting with the E6-sfGFP probe (N=3, mean ± SD). (d) Binding affinities (K_D) for nanobodies as determined in (c).

Figure 3.. Nanobodies reduce colony-forming in HPV+ cells.

Clonogenic assays were performed in (a) CaSki, (b) SiHa, and (c) C33A cells following nanobody transfection. Colonies were stained with crystal violet and counted by Image J software. Colony-forming units were normalized to the mCherry control condition on each plate (N ≥ 3, mean ± SD). Data shown as mean ± SD. Statistical analysis was performed using a one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4.. BioPROTAC^E6 degrades E6, disrupts HPV+ proliferation, induces IFNB1 mRNA and increases p53 activity.

(a) Schematic representing PROTAC^E6 design and structural model illustrating recruitment of E6 to the CUL2 ubiquitin ligase complex (PDB 5N4W). (b) Confocal microscopic images of HEK293T cells transfected with E6-GFP (1.25µg) in combination with VHL, PROTAC^E6, or mutPROTAC^E6 (VHL^{TLK157–159AAA}) (1.25µg each). Cells were imaged 48 hours post-transfection (N=3). Scale bar 400μM (c) Dose-dependent inhibition of CaSki cell proliferation by PROTAC^E6 (0, 161, 193, 279, 334, 401, 482, 578, 694, and 1000 ng of DNA) measured 48 hours post-transfection using the WST-1 assay in 96-well format (N=3, mean ± SD). Data are normalized to control and fitted using Prism GraphPad ([Inhibition] vs. Response). (d) CaSki cells transfected with plasmids encoding Empty control, A2, A5, C11, and PROTAC^E6 were assessed for proliferation 48 hours post-transfection using the WST-1 assay. Data are normalized to Empty control (N=3 or more, data are mean ± SD). (e) CaSki cells were transfected with 2.5μg of plasmids encoding Empty, A2, A5, C11, or PROTAC^E6. At 48 hours, cells were treated with poly(I:C) for 6 hours, and IFNB1 mRNA levels were measured by RT-qPCR and normalized to GAPDH. Data were normalized to Empty control (N=3). The means ± SD for one of three biological representative experiments is shown. (f) Western blot analysis of p53 expression in CaSki cells transfected with Empty, A5, or PROTAC^E6. At 72 hours post-transfection, cells were treated with Nutlin-3e for 3 hours prior to harvest. One representative blot of three is shown (N=3). (g) Quantification of p53 protein levels from (f) by densitometry using ImageJ (N=3, mean ± SD). (h) CaSki cells were transfected with 2.5μg of plasmids encoding Empty, A2, A5, C11, or PROTAC^E6. RT-qPCR analysis of P21 mRNA levels 72 hours after transfection. Data are normalized to Empty control (N=3). The means ± SD for one of three biological representative experiments is shown. Statistical analysis for panels (d), (e), (g), and (h) was performed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5.. PROTAC^E6 mediated reduction in TC-1 tumor burden.

(a) Brightfield microscopic images of TC-1 cells transfected with Empty and PROTAC^E6 and imaged 48 hours post-transfection. (b) Schematic timeline of in vivo treatment protocol. (c) Tumor growth curves for mice treated intratumorally on days 7 and 14 with Empty (N=14), PROTAC^E6 (N=14), and PROTAC^E6 combined with αCD4 and αCD8 (N=8). Tumor volume is presented as mean ± SEM; statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001). (d) Tumor size at endpoint (day 21) was analyzed by one-way ANOVA; data represent mean ± SEM (* p < 0.05, ** p < 0.01).