HPV16 E7 Research Articles

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

Time to take HPV infection in colorectal cancer patients more seriously.

The association between viral infections and colorectal cancer (CRC) remains an enigma in cancer research. Certain types of Human Papillomaviruses (hr-HPVs), known for their oncogenic properties, have been observed in particular CRC biopsies, further adding to the enigma surrounding this association. This cross-sectional study was conducted on 40 confirmed cases of CRC adenocarcinoma. The presence and genotyping of HPV DNA in colorectal fresh tissue and urine samples was assessed using an HPV DNA hybridization kit. A subset of serum samples from both CRC cases and healthy volunteers was randomly chosen and subjected to western blot to investigate the presence of HPV16 E6/E7 oncoproteins carried by exosomes. It was observed that 26/40 HPV-positive CRC patients demonstrated 7 times more chance to develop colorectal cancer when compared to those 8/40 normal tissue (odds ratio [OR] = 7.4; confidence interval [CI] 95% = 0.483156-0.793718; p < 0.001). Of 26 HPV-positive CRC patients, 14 urine samples were also showed HPV DNA positivity (p = 0.013). High-risk HPV16 was the most prevalent genotype detected in both 24/40 tumor and 12/40 urine samples (p < 0.001). The tumor sample of a male was HPV45, while another male's urine sample was HPV31. A female CRC patient had HPV83 in tumor and HPV56 in urine. Here, was the first detection of HPV83 in a CRC patient. Notably among 20 randomly selected serum exosome samples, one serum sample concurrently tested positive for both HPV16 E6 and E7 oncoproteins, and one sample tested positive for HPV16 E7 oncoprotein. High risk HPV DNA detection in CRC urine samples supports non-invasive screening tools. Detection of HPV16 E6 and E7 oncoproteins in exosomes from serum samples shows potential for non-invasive diagnostics. HPV's potential role in CRC development is also underscored. HPV vaccination should be implemented in low- and middle-income countries to prevent cancer.

Circulating tumor HPV DNA in the management of HPV+ oropharyngeal cancer and its correlation with MRI.

First aim was to compare ddPCR assays of ctHPVDNA with p16 IHC and qualitative HPV PCR. Second aim was to carry out longitudinal blood sampling to test for association of ctHPVDNA with histological confirmed recurrence. Third aim was to perform a multidimensional assessment which included: (1) clinical features; (2) ctHPVDNA; (3) MRI-based tumor size measurements of primary tumor (PT) and cervical lymph node metastases (CLNM). Plasma samples were collected before treatment and during follow-up, and ddPCR assay comprising E6 of HPV16 and HPV 33 and HPV 35 was used. Present study was conducted at diagnosis in 117 patients and revealed a ctHPVDNA sensitivity of 100% (95% CI 95.5-100) and a specificity of 94.4 (95% CI 81.3-99.3), positive predictive value (PPV) of 94.4 (95% CI 81.3-99.3), and negative predictive value (NPP) of 100% (95% CI 89.7-100). During follow-up ctHPVDNA had a sensitivity of 100% (95% CI 72.1-100)% and specificity of 98.4% (95% CI 91.7-100)%, PPV% of 90.9% (95% CI 62.3-98.4) and NPV% of 100% (95% CI 94.3-100) for ability to detect recurrence. Correlation between both the CLNM volume and the sum of PT and CLNM volume was observed. ctHPVDNA was superior to p16 in identification of HPV-OPSCC at diagnosis. Introduction of ctHPVDNA, beyond diagnostic setting, represents a great opportunity to improve follow-up protocol of OPSCC patients.

Genetic variability of human papillomavirus type 18 based on E6, E7 and L1 genes in central China

BackgroundHigh-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16.MethodsIn this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively.ResultsA total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes.ConclusionThe sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.

Analysis of human papillomavirus type 16 E4, E5 and L2 gene variations among women with cervical infection in Xinjiang, China

BackgroundThere is a high incidence of cervical cancer in Xinjiang. Genetic variation in human papillomavirus may increase its ability to invade, spread, and escape host immune response.MethodsHPV16 genome was sequenced for 90 positive samples of HPV16 infection. Sequences of the E4, E5 and L2 genes were analysed to reveal sequence variation of HPV16 in Xinjiang and the distribution of variation among the positive samples of HPV16 infection.ResultsEighty-one of the 90 samples of HPV16 infection showed variation in HPV16 E4 gene with 18 nucleotide variation sites, of which 8 sites were synonymous variations and 11 missense variations. 90 samples of HPV16 infection showed variation in HPV16 E5 and L2 genes with 16 nucleotide variation sites (6 synonymous, 11 missense variations) in the E5 gene and 100 nucleotide variation sites in L2 gene (37 synonymous, 67 missense variations). The frequency of HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A was higher in the case groups than in the control groups.ConclusionsPhylogenetic tree analysis showed that 87 samples were European strains, 3 cases were Asian strains, there were no other variations, and G4181A was related to Asian strains. HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A were significantly more frequent in the case groups than in the control groups.

Cationic Liposomes Carrying HPV16 E6-siRNA Inhibit the Proliferation, Migration, and Invasion of Cervical Cancer Cells.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the "hit-and-run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light-induced malignant transformation.

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active site were required for PTPN14 to promote differentiation. Taken together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.

Strong evidence supports the use of estradiol therapy for the treatment of vaginal inflammation: a two-way Mendelian randomization study

ObjectiveNowadays, there has been limited Mendelian randomization (MR) research focusing on the causal relationship between estradiol and vaginitis. Therefore, this study conducted a two-way MR study to clarify the causal effect and related influencing factors between them.MethodsAll genetic datasets were obtained using publicly available summary statistics based on individuals of European ancestry from the IEU GWAS database. MR analysis was performed using MR-Egger, weighted median (WM) and inverse variance weighted (IVW) methods to assess the causal relationship between exposure and outcome and to validate the findings by comprehensively evaluating the effects of pleiotropic effects and outliers.ResultsMR analysis revealed no significant causal relationship between estradiol and vaginitis risk. There was a negative correlation between estradiol and age at menarche (IVW, OR: 0.9996, 95% CI: 0.9992–1.0000, P = 0.0295; WM, OR: 0.9995, 95% CI: 0.9993–0.9998, P = 0.0003), and there was a positive correlation between age at menarche and vaginitis (IVW, OR: 1.5108, 95% CI: 1.1474–2.0930, P = 0.0043; MR-Egger, OR: 2.5575, 95% CI: 1.7664–9.6580, P = 0.0013). Estradiol was negatively correlated with age at menopause (IVW, OR: 0.9872, 95% CI: 0.9786–0.9959, P = 0.0041). However, there was no causal relationship between age at menopause and vaginitis (P > 0.05). In addition, HPV E7 Type 16, HPV E7 Type 18, and Lactobacillus had no direct causal effects on estradiol and vaginitis (P > 0.05). Sensitivity analyses revealed no heterogeneity and horizontal pleiotropy.ConclusionWhen estrogen levels drop, it will lead to a later age of menarche, and a later age of menarche may increase the risk of vaginitis, highlighting that the longer the female reproductive tract receives estrogen stimulation, the stronger the defense ability is formed, and the prevalence of vaginitis is reduced. In conclusion, this study indirectly supports an association between reduced level of estrogen or short time of estrogen stimulation and increased risk of vaginitis.

Quantification of HPV16 E7 Oncoproteins in Urine Specimens from Women with Cervical Intraepithelial Neoplasia.

We present the validity of using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for quantifying high-risk human papillomavirus (HPV) 16 E7 oncoproteins in urine specimens as a noninvasive method of analyzing the oncogenic activity of HPV. Some reports claim that the oncogenic activity of HPV is a more relevant clinical indicator than the presence of HPV DNA for estimating malignant potential. In the present study, urine containing HPV16 and related types were selected by uniplex E6/E7 polymerase chain reaction and classified according to the pathologic diagnosis of cervical intraepithelial neoplasia (CIN) in cervical biopsy specimens. Our ultrasensitive ELISA was able to detect attomole levels of HPV16 E7 oncoproteins, and it detected HPV16-positive SiHa cells at >500 cells/mL without detecting HPV18-positive cells. Our ELISA results showed E7 oncoproteins in 80% (4/5) of urine specimens from women with HPV16-positive CIN1, 71% (5/7) of urine specimens from CIN2 patients, and 38% (3/8) of urine specimens from CIN3 patients. Some urine specimens with undetectable E7 oncoproteins were thought to be negative for live HPV 16-positive cells or in an inactivated state of infection. These results provide the basis for assessing oncogenic activity by quantifying E7 oncoproteins in patient urine.

T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector

We recently developed an immuno-oncotherapy against human papillomavirus (HPV)-induced tumors based on a lentiviral vector encoding the Early E6 and E7 oncoproteins of HPV16 and HPV18 genotypes, namely “Lenti-HPV-07”. The robust and long-lasting anti-tumor efficacy of Lenti-HPV-07 is dependent on CD8+ T-cell induction and remodeling of the tumor microenvironment. Here, we first established that anti-vector immunity induced by Lenti-HPV-07 prime has no impact on the efficacy of a homologous boost to amplify anti-HPV T-cell immunity. To longitudinally monitor the evolution of the T-cell repertoire generated after the prime, homologous or heterologous boost with Lenti-HPV-07, we tracked T-cell clonotypes by deep sequencing of T-Cell Receptor (TCR) variable β and α chain mRNA, applied to whole peripheral blood cells (PBL) and a T cell population specific of an immunodominant E7HPV16 epitope. We observed a hyper-expansion of clonotypes post prime, accompanied by increased frequencies of HPV-07-specific T cells. Additionally, there was a notable diversification of clonotypes post boost in whole PBL, but not in the E7HPV16-specific T cells. We then demonstrated that the effector functions of such Lenti-HPV-07-induced T cells synergize with anti-checkpoint inhibitory treatments by systemic administration of anti-TIM3 or anti-NKG2A monoclonal antibodies. While Lenti-HPV-07 is about to enter a Phase I/IIa clinical trial, these results will help better elucidate its mode of action in immunotherapy against established HPV-mediated malignancies.

Preclinical Immunogenicity and Efficacy Studies for Therapeutic Vaccines for Human Papillomavirus-Type-16-Associated Cancer.

The objective of this study was to conduct preclinical immunogenicity and efficacy studies with several therapeutic vaccines for human papillomavirus (HPV)-16-associated cancers expressing the early antigens E5, E6, and E7 with or without E2. The viral oncoproteins were either expressed by themselves as fusion proteins or the fusion proteins were inserted genetically into herpes simplex virus (HSV)-1 glycoprotein D (gD) which, upon binding to the herpes virus entry mediator (HVEM), inhibits an early T cell checkpoint mediated by the B and T cell mediator (BTLA). This, in turn, lowers the threshold for T cell activation and augments and broadens CD8+ T cell responses to the antigens. The fusion antigens were expressed by chimpanzee adenovirus (AdC) vectors. Expression of the HPV antigens within gD was essential for vaccine immunogenicity and efficacy against challenge with TC-1 cells, which express E7 and E6 of HPV-16 but neither E5 nor E2. Unexpectedly, inclusion of E2 increased both CD8+ T cell responses to the other oncoproteins of HPV-16 and the effectiveness of the vaccines to cause the regression of sizable TC-1 tumors.

Establishment of a prognostic model for hypoxia-associated genes in OPSCC and revelation of intercellular crosstalk.

Hypoxia exerts a profound influence on the tumor microenvironment and immune response, shaping treatment outcomes and prognosis. Utilizing consistency clustering, we discerned two hypoxia subtypes in OPSCC bulk sequencing data from GEO. Key modules within OPSCC were identified through weighted gene correlation network analysis (WGCNA). Core modules underwent CIBERSORT immune infiltration analysis and GSEA functional enrichment. Univariate Cox and LASSO analyses were employed to construct prognostic models for seven hypoxia-related genes. Further investigation into clinical characteristics, the immune microenvironment, and TIDE algorithm prediction for immunotherapy response was conducted in high- and low-risk groups. scRNA-seq data were visually represented through TSNE clustering, employing the scissors algorithm to map hypoxia phenotypes. Interactions among cellular subpopulations were explored using the Cellchat package, with additional assessments of metabolic and transcriptional activities. Integration with clinical data unveiled a prevalence of HPV-positive patients in the low hypoxia and low-risk groups. Immunohistochemical validation demonstrated low TDO2 expression in HPV-positive (P16-positive) patients. Our prediction suggested that HPV16 E7 promotes HIF-1α inhibition, leading to reduced glycolytic activity, ultimately contributing to better prognosis and treatment sensitivity. The scissors algorithm effectively segregated epithelial cells and fibroblasts into distinct clusters based on hypoxia characteristics. Cellular communication analysis illuminated significant crosstalk among hypoxia-associated epithelial, fibroblast, and endothelial cells, potentially fostering tumor proliferation and metastasis.

The utility of HPV16 E6 as a potential biomarker for HPV-related cancers in HIV-infected men who have sex with men.

10526 Background: The burden of HPV-related cancers, including oropharyngeal and anal cancers, poses a significant challenge. Although recent advancements in screening for anal cancer have been made for HIV-infected men who have sex with men (HIV-MSM), establishing effective screening protocols for oropharyngeal cancer remains ongoing. The early oncoprotein HPV16 E6 presents as a promising biomarker for the detection of HPV-associated cancers in this population as well. We investigated the utility of HPV16 E6 in HIV-MSM. Methods: We conducted a retrospective study using an HIV cohort at the AIDS Clinical Center of the National Center for Global Health and Medicine. Participants were all HIV-MSM and included those with HPV-related cancers or precancerous lesions such as oropharyngeal cancer, anal cancer, anal high-grade squamous intraepithelial lesions (aHSIL), and anal low-grade squamous intraepithelial lesions (aLSIL). All cancers and precancerous lesions were histologically confirmed. Frozen plasma samples taken at the time of diagnosis were evaluated. Additionally, when available, samples were retrospectively analyzed up to four years pre- and post-diagnosis. These specimens were sent to the German Cancer Research Center (DKFZ, Heidelberg, Germany) for analysis of antibodies against the early oncoproteins of carcinogenic mucosal HPV types, particularly HPV16 and other high-risk HPVs. Results: One hundred thirty-seven patients living with HIV (PWH) were included in the analysis. The distribution of PWH diagnosed with oropharyngeal cancer, anal cancer, aHSIL, and aLSIL was 6, 14, 96, and 9 respectively. Regarding HPV16 E6 seropositivity, 100% of patients with oropharyngeal cancer tested positive (6/6). Seropositivity rates for anal cancer, aHSIL, and aLSIL were 28.6% (4/14), 12.5% (12/96), and 11.1% (1/9) respectively (Table). Patients with oropharyngeal cancer exhibited the highest titer of HPV16 E6. Among the six patients with oropharyngeal cancer, one demonstrated persistent positivity from four years prior, and four showed positivity from one year prior. Two patients were not assessed as they were newly diagnosed at the time of the study. After chemoradiation treatment, their titers decreased but did not fall below the cutoff level during the follow-up period. In two cases of relapse, the titers, which had initially decreased with chemoradiation, increased again at least one year before the relapse diagnosis. Conclusions: The study demonstrated that screening for HPV16 E6 seropositivity is highly effective for the detection of oropharyngeal cancer in HIV-MSM. This could potentially contribute to early diagnosis and treatment of HPV-related cancers. [Table: see text]

Characterization of novel compounds that directly bind to and inactivate the HPV E6 protein for treatment of HPV-associated cervical and oropharyngeal cancers.

e15122 Background: The human papillomavirus (HPV) E6 protein is necessary for viral genome replication and is always expressed in HPV-induced malignancies. We performed in silico screening of chemical libraries for molecules that bind to a pocket on the cancer associated HPV-16 E6 protein. We focused on the protein-protein interaction of E6 with one of its cellular binding partners, the ubiquitin ligase E6AP. In a trimolecular complex with E6, E6AP ubiquitinates p53, leading to its destruction by the proteasome. We designed, synthesized, and characterized &gt;300 compounds that 1) bind to the E6AP interaction surface of the HPV-16 E6 protein; 2) are armed with a ‘warhead’ to make an irreversible bond with a specific cysteine in this HPV-16 E6 binding pocket; 3) and restore levels of p53 and selectively decrease viability of HPV-16 expressing tumor derived cells. Methods: E6 inhibitor optimization was guided by in silico docking strategies, co-crystallization, and medicinal chemistry, followed by biochemical and cellular assays to iteratively design and synthesize molecules with increased E6 inhibitory activities. Results: We used whole protein mass spectrometry to interrogate covalent bonding to E6, showing irreversible binding to the cysteine in the targeted E6 binding pocket without the other 13 cysteines in E6, with Ki in the range of seconds. Biolayer interferometry and fluorescence polarimetry measured blockade of E6 with E6AP, with IC50 in the range of 400-600 nM. To establish in cell inactivation of E6, human cervical cancer derived SiHa cells that express HPV-16 E6 were incubated with compounds, showing dose dependent increase of p53 levels up to 8-fold after 24 hrs, an effect that was not observed in the HPV-16- cell line RPE-1 expressing wild-type p53. Viability of two cervical (SiHa, CaSki) and two oropharyngeal HPV-16+ cancer cell lines (UM-SC-47, UM-SCC-104) was reduced with an IC50 of ~2-3 µM, with an IC50&gt;30µM in RPE-1 cells and telomerase immortalized human keratinocytes. These in vitro and cell-based experiments revealed dependence on the cysteine residue in the E6AP binding pocket of E6. For in vivo proof-of-concept studies, we developed mouse xenograft models, performed by engrafting SiHa, UM-SC-47 and UM-SCC-104 cancer derived cells subcutaneously into the flank of immunodeficient mice. Lead compounds were administered by intraperitoneal injection and demonstrated nearly complete inhibition (&gt; 70% tumor reduction) in HPV-16 tumor growth compared to vehicle. Anti-tumor activity was not observed with the HPV-negative tumor cell line C33A. Conclusions: HPV-16 is identified in about 50% of all cervical cancers and the majority of head and neck malignancies. Inactivation of the HPV E6 protein represents the opportunity for a novel molecular targeted approach for medical treatment of HPV-16 expressing cancers of the cervix and oropharynx.

Phase II trial assessing safety, efficacy and immune correlates of heterologous prime-boost with pBI-11 (IM) and TA-HPV (IM) plus pembrolizumab for advanced, PD-L1 CPS≥1, hrHPV+ oropharyngeal cancer.

TPS6118 Background: HPV-related OPSCC occurs in younger patients, has a significantly better prognosis, and is most often caused by HPV subtype 16. There is a 90% overall survival for HPV+/p16+ OPSCC (40% for HPV-/p16- OPSCC), which can be cured with multimodality care. For patients with R/M disease, treatment with pembrolizumab (P) +/- chemotherapy is palliative, with a median OS of approximately 12 months for patients with PD-L1 combined positive score (cps) &gt;1 treated with P alone. Heterologous prime boost with DNA priming followed by vaccinia-based boosting against HPV16 viral antigens will be studied using pBI-11 [(DNA vaccine pNGVL4a-SigE7 (detox)/HSP70, HPV16 E7(detox)/ HPV18 E7(detox), HPV16 E6(detox), and HPV18 E6(detox)] followed by TA-HPV (recombinant vaccinia-human papillomavirus (denoted TA-HPV) derived from the Wyeth strain of vaccinia which carries modified E6 and E7 genes from HPV types 16 and 18). This study is approved by the Vanderbilt University IRB and is open accrual (NCT NCT05799144). Methods: Patients with R/M, HPV positive OPSCC without prior therapy for R/M disease, without contraindications to immunotherapy and with a PD-L1 cps &gt; 1 will be screened. There is a 6 patient safety run-in. Treatment with pBI-11 vaccine (2 IM injections on weeks 1 and 4) plus one IM administration of TA-HPV on week 7 will be given in combination with P IV on weeks 1, 4, and 7). Following restaging, all patients will continue (including those with PD) with 3 more cycles of P followed by restaging. The primary clinical outcome is RR to addition of vaccine in the 50% of predicted P non-responders (NR) who proceed to Part II (ie conversion to responders). All responders will continue to receive P until progression. Approximately 54 patients with be enrolled. Two research tumor biopsies and blood draws will be obtained pre- and on treatment (week7-9). Tumor tissue cores will be utilized for immunohistochemical and molecular tests (e.g., expression of immune cell and viral markers, TCR sequencing and total transcripts). Blood with be tested for HPV16/18 E6, E7 antibodies and vaccinia virus neutralizing assays; proliferative responses of peripheral blood lymphocytes to stimulation by HPV16/18 E6 and E7; HPV16/18 E6- and E7-Specific T cells; and HPV DNA in plasma. Data from the 1st four patients enrolled will be presented. Clinical trial information: NCT05799144 .

In silico Analysis of Natural Inhibitors against HPV E6 Protein.

Drug re-purposing is one of the cost-effective methods to establish novel therapeutics against many diseases. Established natural products are collected from databases and used to potentially screen them against HPV E6 protein, a critical viral protein. This study aims to design potential small molecule inhibitors against HPV E6 protein using structure-based approaches. Ten natural anti-cancerous compounds (Apigenin, Baicalein, Baicalin, Ponicidin, Oridonin, Lovastatin, Triterpenoid, Narirutin, Rosmarinic Acid, and Xanthone) were selected by review of the literature. These compounds were screened using Lipinski Rule of Five. Out of ten compounds, seven were found to satisfy Rule of five. Docking of these seven compounds was carried out using AutoDock software and corresponding Molecular Dynamics Simulations were performed by GROMACS. Among the seven compounds docked with the E6 target protein, six compounds showed lesser binding energy than the reference compound, Luteolin. The three-dimensional structures of E6 protein and the corresponding ligand complexes were visualised and analysed using PyMOL whereas the two-dimensional images of protein-ligand interactions were obtained by LigPlot⁺ software to study the specific interactions. ADME analysis using SwissADME software revealed that all the compounds except Rosmarinic acid have good gastrointestinal absorption and solubility characteristics while Xanthone and Lovastatin showed blood brain barrier penetration properties. Considering the binding energy and ADME analysis, Apigenin and Ponicidin are found to be most suitable for de novo</i> designing of potential inhibitors against the HPV16 E6 protein. Further, synthesis and characterization of these potential HPV16 E6 inhibitors will be carried out and their functional evaluation using cell culture-based assays will be undertaken.

Time dependency for HPV ctDNA detection as a prognostic biomarker for anal cancer.

3513 Background: Prior HPV infection is associated with &gt; 90% of anal cancers, a malignancy with rising incidence in the United States. Standard treatment for localized anal cancer is concurrent chemoradiation (cRT). While detection of circulating tumor DNA (ctDNA) within weeks after surgery is linked to recurrence in other solid tumors, the optimal time point for ctDNA detection as a prognostic biomarker following cRT for HPV-associated anogenital cancers is not well characterized. We utilized a novel, highly sensitive HPV ctDNA assay to evaluate clinical outcomes according to HPV ctDNA status among patients with localized anal cancer treated with cRT. Methods: ctDNA was isolated from patients with stages I-III anal cancer treated at our institution with cRT prior to treatment, at the end of treatment (week 5), and at months 3, 6, 9, and 12 after treatment under an IRB-approved protocol. A droplet digital HPV ctDNA PCR assay evaluating HPV E6 and E7 oncogenes for 13 oncogenic HPV types was utilized for quantification of HPV ctDNA. A limit of quantification at 16 HPV copies/mL plasma was set for “HPV ctDNA detection.” Median recurrence-free survival (RFS) according to HPV ctDNA status was estimated via Kaplan-Meier and compared using a log-rank test. Associations between selected clinical factors and recurrence were evaluated with a Chi-square test, with a one-sided p &lt; .05 considered significant. Results: Following cRT, HPV ctDNA was detected in 9/41 (22%), 4/30 (13%), 2/20 (10%), and 0/12 (0%) patients at week 5 and months 3, 6, and 12, respectively. Detection of HPV ctDNA 3 months after cRT was associated with clinical recurrence (100% versus 8%; odds ratio 88, 95% CI 4-2000; p = .006) and inferior RFS (5.9 months vs not reached (NR); hazard ratio (HR) 24, 95% CI 1.2-475; p &lt; .001) relative to HPV ctDNA-negative status. Differences in RFS according to HPV ctDNA status were not observed at week 5/end of treatment (median RFS NR for both; HR 2.4, 95% CI .5-10; p = .15). At month 3, sensitivity and specificity for recurrence according to HPV ctDNA detection were 57% and 100%, respectively, with a PPV and NPV of 100% and 89%, respectively. Baseline clinical stage, T stage, N stage, age, and gender were not associated with clinical recurrence after cRT (p &gt; .25 for all). Conclusions: With a novel, highly sensitive assay, detection of HPV ctDNA at 3 months after cRT was associated with RFS. Further, an HPV ctDNA-positive status outperformed baseline clinical features for prognosticating anal cancer recurrence after cRT in this retrospective series. Future clinical trials should incorporate the 3-month post-treatment time point for identification of patients with HPV-positive anal cancer at elevated risk for recurrence according to HPV ctDNA status. A clinical trial evaluating anti-PD-L1 + anti-TIGIT immunotherapy for patients with HPV-associated cancers with detected HPV ctDNA after cRT is forthcoming at our institution.

A phase 1 dose-escalation and expansion study of CUE-101, given as monotherapy and in combination with pembrolizumab, in patients with recurrent/metastatic HPV16+ head and neck squamous cell cancer (R/M HNSCC).

6004 Background: Immuno-STATs are modular T cell engagers engineered to selectively activate tumor-antigen specific CD8+ T cells via targeted delivery of cytokines. CUE-101, the first Immuno-STAT in clinical trials, is composed of a human leukocyte antigen (HLA) complex, HLA-A*0201, a peptide epitope derived from the HPV16 E7 protein and 4 molecules of attenuated human interleukin-2 (IL-2), to bind, expand, and activate HPV16-specific CD8+ T cells for the treatment of HPV16+ HNSCC. Methods: CUE-101-01 is an ongoing first-in-human study in HLA-A*0201 patients with HPV16+ R/M HNSCC. Escalating doses of CUE-101 monotherapy (0.06 mg/kg to 8 mg/kg) were evaluated in R/M HNSCC refractory to ≥ 1 platinum or checkpoint inhibitor (CPI) based therapy, alone or combined with pembrolizumab (1 mg/kg to 4 mg/kg + 200 mg pembrolizumab) in the first line treatment of PD-L1+ R/M HNSCCs. Enrollment at the recommended phase 2 dose (RP2D) was expanded. Therapy was administered every 3 weeks (Q3W) until disease progression or intolerable toxicity. Safety, PK/PD, and antitumor activity were assessed. Results: Enrollment in both monotherapy and combination cohorts is now completed (N=80 patients, 49 in monotherapy and 31 CUE-101 plus pembrolizumab). Following dose escalation, 4 mg/kg Q3W of CUE-101 was selected for RP2D for both monotherapy and pembrolizumab combination cohorts. At data cut-off, adverse events (AEs) have been manageable and 92% grade ≤2. The most frequent grade 3 AEs reported include lymphocyte count decreased (7.6%), anemia (6.3%), decreased appetite (5.1%) and infusion-related reactions (5.1%). In combination with pembrolizumab no unanticipated significant safety concerns have emerged. Exposure-dependent PD effects of CUE-101 are consistent with IL-2 pharmacology and indicate preferential expansion of CUE-101 on E7-specific T cells. Among the 19 evaluable patients treated with the RP2D of CUE-101 plus pembrolizumab, an ORR of 47% (1 CR, 8 PRs), a Disease Control Rate (ORR + durable SDs) of 74%, and mPFS of 5.8 months [95% CI 2.56; NA] were observed. A median OS has not been reached. Of the 9 patients with confirmed objective responses, all achieved &gt;99% reduction in HPV16 cfDNA in plasma during their treatment course. Among the 19 evaluable monotherapy RP2D patients, 1 PR and 6 durable SD (SD ≥ 12 weeks) and mOS of 20.8 months [95% CI 11.0; NA] were observed. Conclusions: An ORR of 47% and a mPFS of 5.8 months were observed in R/M HNSCC patients treated with CUE-101 4 mg/kg + pembrolizumab as 1L therapy. A median OS of 20.8 was observed in patients treated with CUE-101 monotherapy as post-platinum/CPI therapy. CUE-101 continues to demonstrate safety, tolerability and meaningful clinical benefit in patients with HPV16+ R/M HNSCC. Clinical trial information: NCT03978689 .

Structure-Based Discovery of Potential HPV E6 and EBNA1 Inhibitors: Implications for Cervical Cancer Treatment

Cervical cancer is the fourth most diagnosed cancer and the fourth leading cause of cancer death in women globally. Its onset and progression have been attributed to high-risk human papillomavirus (HPV) types, especially 16 and 18, while the Epstein–Barr virus (EBV) is believed to also significantly contribute to cervical cancer growth. The E6 protein associated with high-risk HPV strains, such as HPV16 and HPV18, is known for its role in promoting cervical cancer and other anogenital cancers. E6 proteins contribute to the malignant transformation of infected cells by targeting and degrading tumor suppressor proteins, especially p53. On the other hand, EBV nuclear antigen 1 (EBNA1) plays a crucial role in the maintenance and replication of the EBV genome in infected cells. EBNA1 is believed to increase HPV E6 and E7 levels, as well as c-MYC, and BIRC5 cellular genes in the HeLa cell line, implying that HPV/EBV co-infection accelerates cervical cancer onset and growth. Thus, the E6 and EBNA1 antigens of HPV and EBV, respectively, are attractive targets for cervical cancer immunotherapy. This study, therefore, virtually screened for potential drug candidates with good binding affinity to all three oncoviral proteins, HPV16 E6, HPV18 E6, and EBNA1. The compounds were further subjected to ADMET profiling, biological activity predictions, molecular dynamics (MD) simulations, and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. A total of six compounds comprising ZINC000013380012, ZINC000070454124, ZINC000014588133, ZINC000085568136, ZINC000095909247, and ZINC000085597263 demonstrated very strong affinity (≤−60 kJ/mol) to the three oncoviral proteins (EBNA1, HPV16 E6, and HPV18 E6) after being subjected to docking, MD, and MM/PBSA. These compounds demonstrated relatively stronger binding than the controls used, inhibitors of EBNA1 (VK-1727) and HPV E6 (baicalein and gossypetin). Biological activity predictions also corroborated their antineoplastic, p53-enhancing, Pin1 inhibitory, and JAK2 inhibitory activities. Further experimental testing is required to validate the ability of the shortlisted compounds to silence the insidious effects of HPV E6 and EBNA1 proteins in cervical cancers.