Is immunotherapy a potential game changer in managing human papillomavirus (HPV) infection and intraepithelial neoplasia?

The International Papillomavirus Conference was held in Washington DC in April 2023 and encompassed wide ranging basic, clinical and public health research relating to animal and human papillomaviruses. This editorial is a personal reflection, it does not attempt to be comprehensive and reports on some key aspects centred on the prospects for immune interventions in prevention and treatment of HPV infections and early precancers with a focus on cervical neoplasia. There is optimism for the future impact of immunotherapy in treating early HPV associated disease. This will depend on developing an appropriate design of vaccines and delivery vehicles which then need to be properly tested in clinical trials that are able to measure a useful clinical endpoint. Thereafter vaccines (prophylactic or therapeutic) still need global access and sufficient uptake to deliver impact and a key and necessary driver is education.

Oropharyngeal Squamous Cell Carcinoma Treatment in the Era of Immune Checkpoint Inhibitors.

While head and neck squamous cell carcinomas (HNSCC) are marginally decreasing due to the reduction in exposure to the major risk factors, tobacco and alcohol, the incidence of high-risk human papillomavirus (HPV)-positive oropharynx squamous cell carcinomas (OPSCC), especially those in the tonsil and base of tongue subsites, are increasing. Patients with the latter are younger, display a longer overall survival, and show a lower recurrence rate after standard-of-care treatment than those with HPV-negative OPSCC. This may reflect an important role for immune surveillance and control during the natural history of the virally driven tumour development. Immune deviation through acquisition of immune-suppressive factors in the tumour microenvironment (TME) is discussed in relation to treatment response. Understanding how the different immune factors are integrated in the TME battleground offers opportunities for identifying prognostic biomarkers as well as novel therapeutic strategies. OPSCC generally receive surgery or radiotherapy for early-stage tumour treatment, but many patients present with locoregionally advanced disease requiring multimodality therapies which can involve considerable complications. This review focuses on the utilization of newly emerged immune checkpoint inhibitors (PD-1/PD-L1 pathway) for treatment of HNSCC, in particular HPV-positive OPSCC, since they could be less toxic and more efficacious. PD-1/PD-L1 expression in the TME has been extensively investigated as a biomarker of patient response but is yet to provide a really effective means for stratification of treatment. Extensive testing of combinations of therapeutic approaches by types and sequencing will fuel the next evolution of treatment for OPSCC.

Harnessing immunity for therapy in human papillomavirus driven cancers.

In persistent high-risk HPV infection, viral gene expression can trigger some important early changes to immune capabilities which act to protect the lesion from immune attack and subsequently promote its growth and ability for sustained immune escape. This includes immune checkpoint-inhibitor ligand expression (e.g. PD-L1) by tumour or associated immune cells that can block any anti-tumour T-cell effectors. While there are encouraging signs of efficacy for cancer immunotherapies including with immune checkpoint inhibitors, therapeutic vaccines and adoptive cell therapies, overall response and survival rates remain relatively low. HPV oncogene vaccination has shown some useful efficacy in treatment of patients with high-grade lesions but was unable to control later stage cancers. To maximally exploit anti-tumour immune responses, the suppressive factors associated with HPV carcinogenesis must be countered. Importantly, a combination of chemotherapy, reducing immunosuppressive myeloid cells, with therapeutic HPV vaccination significantly improves impact on cancer treatment. Many clinical trials are investigating checkpoint inhibitor treatments in HPV associated cancers but response rates are limited; combination with vaccination is being tested. Further investigation of how chemo- and/or radio-therapy can influence the recovery of effective anti-tumour immunity is warranted. Understanding how to optimally deploy and sequence conventional and immunotherapies is the challenge.

Spatial proximity between T and PD-L1 expressing cells as a prognostic biomarker for oropharyngeal squamous cell carcinoma.

BACKGROUND: Fulfilling the promise of cancer immunotherapy requires novel predictive biomarkers to characterise the host immune microenvironment. Deciphering the complexity of immune cell interactions requires an automated multiplex approach to histological analysis of tumour sections. We tested a new automatic approach to select tissue and quantify the frequencies of cell-cell spatial interactions occurring in the PD1/PD-L1 pathway, hypothesised to reflect immune escape in oropharyngeal squamous cell carcinoma (OPSCC). METHODS: Single sections of diagnostic biopsies from 72 OPSCC patients were stained using multiplex immunofluorescence (CD8, PD1, PD-L1, CD68). Following multispectral scanning and automated regions-of-interest selection, the Hypothesised Interaction Distribution (HID) method quantified spatial proximity between cells. Method applicability was tested by investigating the prognostic significance of co-localised cells (within 30 µm) in patients stratified by HPV status. RESULTS: High frequencies of proximal CD8+ and PD-L1+ (HR 2.95, p = 0.025) and PD1+ and PD-L1+ (HR 2.64, p = 0.042) cells were prognostic for poor overall survival in patients with HPV negative OPSCC (n = 31). CONCLUSION: The HID method can quantify spatial interactions considered to reflect immune escape and generate prognostic information in OPSCC. The new automated approach is ready to test in additional cohorts and its applicability should be explored in research and clinical studies.

Correction to: Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer.

The article Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer, written by Y. Louise Wan, Puja Sapra, James Bolton, Jia Xin Chua, Lindy G. Durrant and Peter L. Stern, was originally published under a CC BY-NC 4.0 license, but has now been made available under a CC BY 4.0 license.

Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer.

BACKGROUND: Recurrence occurs in over 75% of women with epithelial ovarian cancer despite optimal treatment. Selectively killing tumour cells thought to initiate relapse using an antibody-drug conjugate could prolong progression-free survival and offer an improved side-effect profile. A1mcMMAF is an antibody-drug conjugate designed to target cells expressing the tumour-associated antigen 5T4. It has shown to be efficacious in various cell line models and have a greater impact when combined with routine chemotherapeutic regimes. OBJECTIVES: This study aims to explore the potential for the use of a 5T4 antibody-drug conjugate in women with ovarian cancer both as a monotherapy and in combination with platinum-based chemotherapy. METHODS: Immunohistochemical analysis was used to assess 5T4 expression in tumours from patients with ovarian cancer. Effectiveness of A1mcMMAF therapy as a single agent and in combination with carboplatin was assessed in vitro in the ovarian cancer cell line SKOV3 and confirmed in vivo using a serial bioluminescence assay in a SKOV3 xenograft model of ovarian cancer. RESULTS: 5T4 is confirmed as suitably expressed in epithelial ovarian cancers prior to adjuvant therapy and is an independent predictor of poor survival. A1mcMMAF showed specific activity, both in vitro and in vivo, against SKOV3 ovarian cancer cells. When used in combination with carboplatin, in vivo tumour growth was inhibited resulting in prolonged survival in a SKOV3 xenograft model. CONCLUSIONS: These data support further investigation of A1mcMMAF in combination with platinum-based chemotherapy in ovarian and other cancer treatments.

Opportunities to improve immune-based prevention of HPV-associated cancers.

Immunization of adolescent girls with VLP vaccines, made of L1 proteins from the most medically significant high risk HPV types, is a major strategy for prevention of cervical cancer plus other HPV-associated cancers. Maximal population impact, including through herd immunity, requires high vaccination coverage. However, protection of unvaccinated women requires secondary prevention through cytology screening. Unfortunately in countries with the highest incidence/mortality due to cervical cancer HPV vaccination (or cytology screening) is not sufficiently available. Vaccination programme costs and a lack of accessibility of the populations for immunization remain significant hurdles. Several approaches could increase effective implementation of HPV vaccination. 1) Use of a single immunization of the current VLP vaccines. 2) Vaccination bundled with other paediatric vaccines with lower dosage to facilitate delivery, improve coverage and reduce costs through established logistics. 3) Local manufacture with lower cost systems (e.g. bacteria) for VLP or capsomer based vaccine production and utilization of additional protective epitopes (e.g L2) for increasing breadth of protection. However, all the latter need appropriate clinical validation. Gender neutral vaccination and extending routine vaccination strategies to women up to age 30 years in combination with at least one HPV screening test can also hasten impact on cancer incidence.

Cancer stem cell mobilization and therapeutic targeting of the 5T4 oncofetal antigen.

Cancer stem cells (CSCs) can act as the cellular drivers of tumors harnessing stem cell properties that contribute to tumorigenesis either as founder elements or by the gain of stem cell traits by the malignant cells. Thus, CSCs can self-renew and generate the cellular heterogeneity of tumors including a hierarchical organization similar to the normal tissue. While the principle tumor growth contribution is often from the non-CSC components, it is the ability of small numbers of CSCs to avoid the effects of therapeutic strategies that can contribute to recurrence after treatment. However, identifying and characterizing CSCs for therapeutic targeting is made more challenging by their cellular potency being influenced by a particular tissue niche or by the capacity of more committed cells to regain stem cell functions. This review discusses the properties of CSCs including the limitations of the available cell surface markers, the assays that document tumor initiation and clonogenicity, the roles of epithelial mesenchymal transition and molecular pathways such as Notch, Wnt, Hippo and Hedgehog. The ability to target and eliminate CSCs is thought to be critical in the search for curative cancer treatments. The oncofetal tumor-associated antigen 5T4 (TBGP) has been linked with CSC properties in several different malignancies. 5T4 has functional attributes that are relevant to the spread of tumors including through EMT, CXCR4/CXCL12, Wnt, and Hippo pathways which may all contribute through the mobilization of CSCs. There are several different immunotherapies targeting 5T4 in development including antibody-drug conjugates, antibody-targeted bacterial super-antigens, a Modified Vaccinia Ankara-basedvaccine and 5T4-directed chimeric antigen receptor T-cells. These immune therapies would have the advantage of targeting both the bulk tumor as well as mobilized CSC populations.

Opportunities and challenges for human papillomavirus vaccination in cancer.

The discovery of genotype 16 as the prototype oncogenic human papillomavirus (HPV) initiated a quarter century of laboratory and epidemiological studies that demonstrated their necessary, but not sufficient, aetiological role in cervical and several other anogenital and oropharyngeal cancers. Early virus-induced immune deviation can lead to persistent subclinical infection that brings the risk of progression to cancer. Effective secondary prevention of cervical cancer through cytological and/or HPV screening depends on regular and widespread use in the general population, but coverage is inadequate in low-resource settings. The discovery that the major capsid antigen L1 could self-assemble into empty virus-like particles (VLPs) that are both highly immunogenic and protective led to the licensure of several prophylactic VLP-based HPV vaccines for the prevention of cervical cancer. The implementation of vaccination programmes in adolescent females is underway in many countries, but their impact critically depends on the population coverage and is improved by herd immunity. This Review considers how our expanding knowledge of the virology and immunology of HPV infection can be exploited to improve vaccine technologies and delivery of such preventive strategies to maximize reductions in HPV-associated disease, including incorporation of an HPV vaccine covering oncogenic types within a standard multitarget paediatric vaccine.

The failure of radical treatments to cure cancer: can less deliver more?

All too often attempts to deliver improved cancer cure rates by increasing the dose of a particular treatment are not successful enough to justify the accompanying increase in toxicity and reduction in quality of life suffered by a significant number of patients. In part, this drive for using higher levels of treatment derives from the nature of the process for testing and incorporation of new protocols. Indeed, new treatment regimens must now consider the key role of immunity in cancer control, a component that has been largely ignored until very recently. The recognition that some drugs developed for cytotoxicity at higher doses can display alternative anticancer activities at lower doses including through modulation of immune responses is prompting a significant re-evaluation of treatment protocol development. Given that tumours are remarkably heterogeneous and with inherent genetic instability it is probably only the adaptive immune response with its flexibility and extensive repertoire that can rise to the challenge of effecting significant control and ultimately elimination of a patient's cancer. This article discusses some of the elements that have limited higher levels of treatment outcomes and where too much proved less effective. We explore observations that less can often be as effective, if not more effective especially with some chemotherapy regimens, and discuss how this can be exploited in combination with immunotherapies to deliver nontoxic improved tumour responses.

Human papillomavirus-driven immune deviation: challenge and novel opportunity for immunotherapy.

It is now recognized that the immune system can be a key component of restraint and control during the neoplastic process. Human papillomavirus (HPV)-associated cancers of the anogenital tract and oropharynx represent a significant clinical problem but there is a clear opportunity for immune targeting of the viral oncogene expression that drives cancer development. However, high-risk HPV infection of the target epithelium and the expression of the E6/E7 oncogenes can lead to early compromise of the innate immune system (loss of antigen-presenting cells) facilitating viral persistence and increased risk of cancer. In these circumstances, a succession of interacting and self-reinforcing events mediated through modulation of different immune receptors, chemokine and cytokine responses (CCL20; CCL2; CCR2; IL-6; CCR7; IL-12) further promote the generation of an immune suppressive microenvironment [increased levels of Tregs, Th17, myeloid-derived suppressor cells (MDSCs) and PD-L1]. The overexpression of E6/E7 expression also compromises the ability to repair cellular DNA, leading to genomic instability, with the acquisition of genetic changes providing for the selection of advantaged cancer cells including additional strategies for immune escape. Therapeutic vaccines targeting the HPV oncogenes have shown some encouraging success in some recent early-phase clinical trials tested in patients with HPV-associated high-grade anogenital lesions. A significant hurdle to success in more advanced disease will be the local and systemic immune suppressive factors. Interventions targeting the different immunosuppressive components can provide opportunity to release existing or generate new and effective antitumour immunity. Treatments that alter the protumour inflammatory environment including toll-like receptor stimulation, inhibition of IL-6-related pathways, immune-checkpoint inhibition, direct modulation of MDSCs, Tregs and macrophages could all be useful in combination with therapeutic HPV vaccination. Future progress in delivering successful immunotherapy will depend on the configuration of treatment protocols in an insightful and timely combination.

Targeting the 5T4 oncofetal glycoprotein with an antibody drug conjugate (A1mcMMAF) improves survival in patient-derived xenograft models of acute lymphoblastic leukemia.

Outcome in childhood acute lymphoblastic leukemia is prognosticated from levels of minimal residual disease after remission induction therapy. Higher levels of minimal residual disease are associated with inferior results even with intensification of therapy, thus suggesting that identification and targeting of minimal residual disease cells could be a therapeutic strategy. Here we identify high expression of 5T4 in subclonal populations of patient-derived xenografts from patients with high, post-induction levels of minimal residual disease. 5T4-positive cells showed preferential ability to overcome the NOD-scidIL2Rγnull mouse xenograft barrier, migrated in vitro on a CXCL12 gradient, preferentially localized to bone marrow in vivo and displayed the ability to reconstitute the original clonal composition on limited dilution engraftment. Treatment with A1mcMMAF (a 5T4-antibody drug conjugate) significantly improved survival without overt toxicity in mice engrafted with a 5T4-positive acute lymphoblastic leukemia cell line. Mice engrafted with 5T4-positive patient-derived xenograft cells were treated with combination chemotherapy or dexamethasone alone and then given A1mcMMAF in the minimal residual disease setting. Combination chemotherapy was toxic to NOD-scidIL2Rγnull mice. While dexamethasone or A1mcMMAF alone improved outcomes, the sequential administration of dexamethasone and A1mcMMAF significantly improved survival (P=0.0006) over either monotherapy. These data show that specifically targeting minimal residual disease cells improved outcomes and support further investigation of A1mcMMAF in patients with high-risk B-cell precursor acute lymphoblastic leukemia identified by 5T4 expression at diagnosis.

Distinct patterns of infiltrating CD8+ T cells in HPV+ and CD68 macrophages in HPV- oropharyngeal squamous cell carcinomas are associated with better clinical outcome but PD-L1 expression is not prognostic.

Immunotherapies are beginning to revolutionise treatment paradigms in oncology with monoclonal antibodies (mAb) targeting T-cell co-inhibitory (e.g. PD-1/PD-L1) and co-stimulatory pathways (e.g. CTLA-4/CD28) demonstrating clinical utility. Some clinical studies demonstrate that responsiveness to PD-1/PD-L1 mAb therapy is greater in patients with expression of PD-L1 in the tumour microenvironment. However, robust responses have also been observed in patients with low or absent expression of PD-L1. Using multiplex immuno-fluorescent labelling we sought to determine how infiltration of tumours by CD8+ T-cells, their expression of PD-1, and the expression of PD-L1 on both tumours and CD68 cells (macrophages) correlated with HPV status and outcome in a cohort of 124 oropharyngeal squamous cell carcinomas (OPSCC).

5T4 oncofoetal antigen: an attractive target for immune intervention in cancer.

The natural history of a patient's cancer is often characterised by genetic diversity and sequential sweeps of clonal dominance. It is therefore not surprising that identifying the most appropriate tumour-associated antigen for targeted intervention is challenging. The 5T4 oncofoetal antigen was identified by searching for surface molecules shared between human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host. The 5T4 protein is expressed by many different cancers but rarely in normal adult tissues. 5T4 molecules are 72 kD, heavily N-glycosylated proteins with several leucine-rich repeats which are often associated with protein-protein interactions. 5T4 expression is associated with the directional movement of cells through epithelial mesenchymal transition, potentiation of CXCL12/CXCR4 chemotaxis and inhibition of canonical Wnt/beta-catenin while favouring non-canonical pathway signalling; all processes which help drive the spread of cancer cells. The selective pattern of 5T4 tumour expression, association with a tumour-initiating phenotype plus a mechanistic involvement with cancer spread have underwritten the clinical development of different immunotherapeutic strategies including a vaccine, a tumour-targeted superantigen and an antibody drug conjugate. In addition, a chimeric antigen receptor T cell approach targeting 5T4 expressing tumour cells is in pre-clinical development. A key challenge will include how best to combine each 5T4 targeted immunotherapy with the most appropriate standard of care treatment (or adjunct therapy) to maximise the recovery of immune control and ultimately eliminate the tumour.

Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies.

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies.

Global gene expression in pseudomyxoma peritonei, with parallel development of two immortalized cell lines.

Pseudomyxoma peritonei (PMP) is a rare tumor of appendiceal origin. Treatment is major cytoreductive surgery but morbidity is high. PMP is considered chemo-resistant; its molecular biology is understudied; and presently, there is no platform for pre-clinical drug testing. Here, we performed exon array analysis from laser micro-dissected PMP tissue and normal colonic epithelia. The array analysis identified 27 up-regulated and 34 down-regulated genes: candidate up-regulated genes included SLC16A4, DSC3, Aldolase B, EPHX4, and ARHGAP24; candidate down-regulated genes were MS4A12, TMIGD1 and Caspase-5. We confirmed differential expression of the candidate genes and their protein products using in-situ hybridization and immuno-histochemistry. In parallel, we established two primary PMP cell lines, N14A and N15A, and immortalized with an SV40 T-antigen lentiviral vector. We cross-checked for expression of the candidate genes (from the array analyses) using qPCR in the cell lines and demonstrated that the gene profiles were distinct from those of colorectal tumor libraries and commonly used colon cell lines. N14A and N15A were responsiveness to mitomycin and oxaliplatin. This study characterizes global gene expression in PMP, and the parallel development of the first immortalized PMP cell lines; fit for pre-clinical testing and PMP oncogene discovery.

Understanding and exploiting 5T4 oncofoetal glycoprotein expression.

Oncofoetal antigens are present during foetal development with generally limited expression in the adult but are upregulated in cancer. These molecules can sometimes be used to diagnose or follow treatment of tumours or as a target for different immunotherapies. The 5T4 oncofoetal glycoprotein was identified by searching for shared surface molecules of human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host, potentially influencing growth, invasion or altered immune surveillance of the host. 5T4 tumour selective expression has stimulated the development of 5T4 vaccine, 5T4 antibody targeted-superantigen and 5T4 antibody-drug therapies through preclinical and into clinical studies. It is now apparent that 5T4 expression is a marker of the use (or not) of several cellular pathways relevant to tumour growth and spread. Thus 5T4 expression is mechanistically associated with the directional movement of cells through epithelial mesenchymal transition, facilitation of CXCL12/CXCR4 chemotaxis, blocking of canonical Wnt/beta-catenin while favouring non-canonical pathway signalling. These processes are highly regulated in development and in normal adult tissues but can contribute to the spread of cancer cells. Understanding the differential impact of these pathways marked by 5T4 can potentially improve existing, or aid development of novel cancer treatment strategies.