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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., (Copyright © 2021 The Author. Published by Elsevier B.V. All rights reserved.)

Objective: The goal of a vaccine is to prime the immune response so the immune memory can facilitate a rapid response to adequately control the pathogen on natural infection and prevent disease manifestation. This article reviews the main elements that provide for the development of safe and effective vaccines., Data Sources: Literature covering target pathogen epidemiology, the key aspects of the functioning immune response underwriting target antigen selection, optimal vaccine formulation, preclinical and clinical trial studies necessary to deliver safe and efficacious immunization., Study Selections: Whole live, inactivated, attenuated, or partial fractionated organism-based vaccines are discussed in respect of the balance of reactogenicity and immunogenicity. The use of adjuvants to compensate for reduced immunogenicity is described. The requirements from preclinical studies, including establishing a proof of principle in animal models, the design of clinical trials with healthy volunteers that lead to licensure and beyond are reviewed., Results: The 3 vaccine development phases, preclinical, clinical, and post-licensure, integrate the requirements to ensure safety, immunogenicity, and efficacy in the final licensed product. Continuing monitoring of efficacy and safety in the immunized populations is essential to sustain confidence in vaccination programs., Conclusion: In an era of increasing vaccine hesitancy, the need for a better and widespread understanding of how immunization acts to counteract the continuing and changing risks from the pathogenic world is required. This demands a societal responsibility for obligate education on the benefits of vaccination, which as a medical intervention has saved more lives than any other procedure., (Copyright © 2020 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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.

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.

Background: Multicellular taste buds located within taste papillae on the tongue mediate taste sensation. In taste papillae, taste bud cells (TBCs), such as taste receptor cells and taste precursor cells, and the surrounding lingual epithelium including epithelial progenitors (also called taste stem/progenitor cells) are maintained by continuous cell turnover throughout life. However, it remains unknown how the cells constituting taste buds proliferate and differentiate to maintain taste bud tissue. Based on in situ hybridization (ISH) screening, we demonstrated that the oncofetal antigen 5T4 (also known as trophoblast glycoprotein: TPBG ) gene is expressed in the adult mouse tongue. Results: In immunohistochemistry of coronal tongue sections, 5T4 protein was detected at a low level exclusively in the basal part of the lingual epithelium in developing and adult mice, and at a high level particularly in foliate papillae and circumvallate papillae (CVPs). Furthermore, immunohistochemistry of the basal part of CVPs indicated that the proliferation marker PCNA (proliferating cell nuclear antigen) co-localized with 5T4. 5T4 was strongly expressed in Krt5 + epithelial progenitors and Shh + taste precursor cells, but weakly in mature taste receptor cells. The number of proliferating cells in the CVP was higher in 5T4- knockout mice than in wild-type (WT) mice, while neither cell differentiation nor the size of taste buds differed between these two groups of mice. Notably, X-ray irradiation enhanced cell proliferation more in 5T4 -knockout mice than in WT mice. Conclusion: Our results suggest that 5T4, expressed in epithelial progenitors (taste stem/progenitor cells), and taste precursor cells, may influence the maintenance of taste papillae under both normal and injury conditions.

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., (Copyright © 2019. Published by Elsevier B.V.)

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., Competing Interests: Conflict of interest statement: RH is a full-time employee of Oxford BioMedica who are developing 5T4-targeted therapies. PLS has been a scientific advisor for GSK, Alligator Biosciences, NeoTx Therapeutics and Oxford BioMedica.

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., Competing Interests: Conflict of interest statement: AGD receives core funding from the ICVI charity, project grants from Celgene, LDNPharma and Jay Pharma. PLS has acted as a scientific advisor for GlaxoSmithKIne, Alligator Biosciences, NeoTx Therapeutics and Oxford BioMedica.

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.

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- scid IL2Rγ null 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- scid IL2Rγ null 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., (Copyright© Ferrata Storti Foundation.)

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., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest.

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.

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).

Unlabelled: Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. The OB possesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors., Significance Statement: Neuronal circuits in the brain include glutamatergic principal neurons and GABAergic interneurons. Although the latter is a minority cell type, they are vital for normal brain function because they regulate the activity of principal neurons. If interneuron function is impaired, brain function may be damaged, leading to behavior disorder. The olfactory bulb (OB) possesses various types of interneurons, including granule cells (GCs); however, the contribution that each type of interneuron makes to the control of olfactory behavior remains unknown. Here, we analyzed electrophysiologically and behaviorally the function of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic branching in OB GCs. We found that, among the various types of OB interneuron, the 5T4 GC subtype is required for odor detection and odor discrimination behaviors., (Copyright © 2016 the authors 0270-6474/16/368211-18$15.00/0.)

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.

Background: The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8(+) T cells recognizing h5T4 can be generated in the absence of CD4(+) T cells from peripheral blood lymphocytes of human healthy individuals., Results: We report the existence and expansion of human CD4(+) T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25(+) cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4(+) T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4(+) T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis., Conclusion: Our data show that CD4(+) T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4(+) T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses.

Purpose: CD4(+)CD25(+) regulatory T (T(reg)) cells are present in increased numbers in patients with advanced cancer and CD25(+) T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25(+) depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets., Patients and Methods: Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25(+) cells using CliniMACS System then re-infused into the patient., Results: Efficient CD25(+) depletion from all leukapheresis products was achieved and 0.55-5.87 x 10(7)/kg CD3(+) cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25(+) subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T(reg) cells., Conclusions: Given the transient nature of the reduction in CD25(+) subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T(reg) cell levels are depleted.

The 5T4 oncofetal antigen is expressed by a wide variety of human carcinomas, including colorectal, ovarian and gastric carcinomas. The restricted expression of 5T4 on tumor tissues as well as its implication in tumor progression and bad prognosis makes 5T4 a promising new candidate for immunotherapy. An MVA vaccine encoding 5T4 antigen has been successfully evaluated in preclinical studies in a murine tumor model. Here, we report the generation of human CD8 T cells specific for the 5T4 antigen by stimulation with autologous monocyte derived DC infected with a replication defective adenovirus encoding the 5T4 cDNA (Ad5T4). Analysis of several donors confirms a repertoire of such CD8 responses. In a parallel approach, incorporating the results of proteasome-mediated digestion of 5T4 derived 35-mer peptides and the potential high affinity epitopes predicted by a computer-based algorithm, we identified 8 putative HLA-A*0201-presented CD8 MHC class I epitopes of 5T4 antigen. Two of these generated specific CD8 T cells after restimulation with peptide loaded autologous DC and assay by cytotoxicity and IFN gamma ELISPOT. Moreover these particular peptide generated T cells recognized naturally 5T4 positive tumor cells only if they expressed HLA-A*0201 as judged by IFN gamma ELISPOT or ELISA. Also, HLA-A*0201 CD8 T cells recognized these peptides in a DC-Ad5T4 polyclonal response. In conclusion, there is a repertoire of CD8 T cell recognition of 5T4 in normal human donors and some candidate HLA-A*0201 epitopes have been identified., (Copyright 2006 Wiley-Liss, Inc.)

The 5T4 oncofoetal antigen is a cell surface glycoprotein that is transiently expressed during mouse ES cell differentiation and correlates with decreased pluripotency of such cells. We show that 5T4 antigen is transiently unregulated during HES4 and H1 human ES cell differentiation and its expression correlates with loss of the pluripotent markers OCT-4 and Tra-1-60 and upregulation of transcript markers associated with the three primary germ layers. To confirm that absence of cell surface 5T4 antigen represents a pluripotent hES cell phenotype, we performed mechanical transfer of either 5T4-ve or 5T4+ve HES4 colonies identified using live cell staining. 5T4-ve transfers maintained expression of OCT-4 in over 90% of resultant colonies, whereas 5T4+ve transfers exhibited significantly lower numbers of OCT-4-expressing colonies (92 +/- 1.4 vs. 2.9 +/- 2.0%). Interestingly, low cell density 5T4-ve colony transfers exhibited increased numbers of OCT-4-expressing colonies compared to large 5T4-ve transfers (92 +/- 1.4 vs. 63.2 +/- 1.9%). 5T4-ve and 5T4+ve HES4 and H1 ES cell lines expressed markers representative of neuroectoderm lineages, and we assessed the formation of neural lineages from these phenotypes in serum-containing medium and N2B27 medium. Expression of 5T4 was found to be inversely related to the yield of tyrosine-hydroxylase (TH+)-expressing neurons in N2B27 medium, with additional mesoderm and endoderm transcript markers detected. Homogeneous glial cell populations were derived from low cell density 5T4-ve colony transfers cultured in serum-containing medium, with TH+ neuronal formation inhibited in a cell-density-dependent manner. We conclude that the 5T4 antigen is a transient marker of hES cell differentiation and that 5T4 phenotype, colony seeding density and culture conditions significantly influence the maintenance of pluripotent hES cells and their differentiation to neural lineages.

This review discusses: (1) immune mechanisms relevant to the natural control of a human papillomavirus (HPV) infection; (2) viral strategies to evade or subvert immune attack; (3) the significance of immune escape as a feature of the evolution of invasive cancer; (4) vaccine strategies for prevention and/or therapy. HPV infection and associated malignancy can induce humoral and cellular immunity to capsid and oncogene viral proteins, but it is not always clear whether such responses are a consequence of the disease rather than the resolving factor(s). Prophylactic strategies are utilising virus-like particles (VLP) composed of the L1 viral capsid protein to induce neutralising antibodies, while therapeutic approaches are aimed at generating specific T cells targeted at the viral E6 and/or E7 oncogenes. Thus far, HPV VLP vaccines have proved clinically efficacious in the early clinical trials to prevent infection. Different types of therapeutic vaccines including peptide, protein, DNA or viral vector encoded have proved safe and immunogenic in patients, although there is often no correlation with clinical outcome. Understanding the equilibrium between viral and immunological factors will be important in providing the appropriate tools to evoke effective prophylactic and therapeutic immunity. It seems likely that combined prophylactic and therapeutic vaccine approaches could offer the best prospect for any significant reduction in death from cervical cancer worldwide.

Infection by high-risk HPV (human papillomavirus) is supposed to be the primary cause of cervical cancer. The HPV E2 protein (E2) is a DNA-binding protein that regulates viral gene expression and is required for efficient viral replication. Overexpression of the E2 protein in cervical cancer cells can induce growth arrest and/or apoptotic cell death, suggesting that E2 might be useful in the treatment of this disease. In the present study, we show that VP22 (herpes simplex virus VP22 protein) can be used to deliver E2 to target cells. VP22-E2 fusion proteins induce apoptosis in transiently transfected HPV-transformed cervical carcinoma cell lines. However, VP22-E2 fusion proteins do not kill COS-7 cells, probably because these cells constitutively express the simian-virus-40 T antigen and this protein sequesters the tumour suppressor protein p53. When COS-7 cells producing VP22-E2 are seeded into cultures of HPV-transformed cells, VP22-E2 enters the non-producing cells and induces apoptosis. VP22-E2 proteins produced in bacterial cells can also enter cervical cancer cells and induce apoptosis in a dose-dependent manner. Our results suggest that local delivery of VP22-E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass (ICM)/epiblast of preimplantation embryos and are widely used in cell differentiation studies. We have previously observed differences in transcript and antigen expression following differentiation of ES cells lines in vitro. We have investigated this further by comparing the differentiation characteristics of five independently derived ES cell lines cultured and differentiated under defined conditions. Undifferentiated ES cell lines exhibited similar morphology and antigen/transcript marker expression. However, upon differentiation in monolayer culture by LIF withdrawal, only two of the lines expressed similar germ layer transcript profiles, and these were significantly altered compared to differentiation in serum-supplemented media. Neurofilament-68k was the only transcript marker common to all cell lines, however, induction of neuroectoderm lineages using 1 microM all-trans retinoic acid (RA) resulted in significant variations in cell number and morphology between the lines. Furthermore, neurons were only formed from clones of the two cell lines that exhibited similar transcript profiles, although the morphology was different between the two. We conclude that the independent ES cell lines in this study differ in their response to alterations in culture conditions in vitro, and the use of an appropriate cell line enables relatively homogeneous neuronal populations to be achieved in monolayer culture under defined conditions.

This study assessed the immunological and clinical responses of women with human papillomavirus (HPV) 16-associated high-grade vulval intraepithelial neoplasia (VIN) vaccinated with TA-HPV, a recombinant vaccinia virus encoding modified HPV 16 and 18 E6 and E7. Eighteen women with HPV 16-positive high-grade VIN were vaccinated with TA-HPV. The extent of their baseline disease was compared after 24 weeks by lesion measurements and histological analysis. Viral load was assessed pre- and postvaccination by real time PCR. Cell-mediated immunity to HPV 16 E6 and/or E7 peptides (HLA-A2 epitopes) or vaccinia-infected cell lysates was determined by IFN-gamma enzyme-linked immunospot (ELISPOT) and T cell proliferation using an HPV 16 L2E6E7 fusion protein. Antibodies were measured by ELISA using vaccinia-infected cell lysates or HPV 16 and 18 E6 and E7 glutathione S-transferase-fusion proteins. Lesion-infiltrating CD4(+), CD8(+), CD1a(+), and CD68(+) immune cells were assessed by immunohistochemistry. The single vaccination with TA-HPV was well tolerated, and all patients showed an increased ELISPOT and/or antibody response to vaccinia. There were significant differences in HPV-16 E7-specific ELISPOT and L2E6E7 proliferative responses in the patients at one or more time points postvaccination as compared with the prevaccination status; two patients showed transient increased antibody responses. Overall, 13 women showed an increased HPV 16-specific immune response by one or more methodologies after immunization. Eight patients demonstrated a reduction in lesion diameter of at least 50% and a further four patients showed significant symptom relief. Viral load was reduced or cleared in six of eight lesion responders but also in six of ten nonresponders. Before vaccination, clinical responders had significantly higher levels of lesion-associated CD4(+), CD8(+), and CD1a(+)-immune cells than nonresponders. There were no differences in CD68 (macrophages) between responders and nonresponders before or after vaccination. Nonresponders did show a significant increase in CD4(+)- and CD8(+)- but not CD1a(+)-immune cells postvaccination but at lower levels overall than responder patients. Local immune infiltration may be a critical factor in potential responsiveness to vaccine therapy in HPV-associated neoplasia and should be carefully monitored in future placebo-controlled trials of immunotherapy for VIN.

Polymorphisms in human leukocyte antigen (HLA) genes have been implicated in the risk for developing human papillomavirus (HPV)-associated cervical neoplasia. By comparison with local cadaver controls typed for HLA class I (n = 946) and II (n = 144) antigens, HPV-16-positive high grade vulval intraepithelial neoplasia patients (n = 42) showed significantly different frequencies of HLA-A2 [odds ratio (OR), 2.1; confidence interval (CI), 1.4-3.9], HLA-B7 (OR, 2.6; CI, 1.4-4.7), HLA-DRB1*01(01/02/04) (OR, 0.1; CI, 0.03-0.5), HLA-DRB1*11 (OR, 3.3; CI, 1.4-7.1), HLA-DRB1*13 (OR, 0), HLA-DQB1*05 (OR, 0.2; CI, 0.05-0.6), and HLA-DQB1*03032 (OR, 4.6; CI, 1.5-14.0). With the exception of HLA-B7 and HLA-DRB1*11, these significant differences were also seen comparative to local HPV-16-positive cervical carcinoma patients (n = 114), suggesting a specific immunogenetic contribution that is independent of HPV-16 infection in high-grade vulval intraepithelial neoplasia. Such factors are important to the development of HPV vaccines for treatment of cervical and vulval neoplasia.

Purpose: Cervical cancer, the second most common malignancy in women worldwide, is almost invariably associated with infection by human papillomavirus (HPV). HPV-16 or -18 is commonly present in 70% of cervical cancers. HPV-positive tumor cells present antigens of the viral protein in the context of human leukocyte antigen (HLA) class I that can be recognized by CTLs. We have conducted a study in patients with early-stage cervical cancer to assess the safety and immunological effects of vaccination with TA-HPV, a live recombinant vaccinia virus expressing modified forms of the HPV-16 and -18 E6 and E7 proteins., Experimental Design: Twenty-nine patients with clinical International Federation of Gynecologists and Obstetricians (FIGO) stage Ib or IIa cervical cancer were given two vaccinations with TA-HPV at least 4 weeks apart, starting 2 weeks before radical hysterectomy. Patients were monitored closely for side effects of the vaccination. Serial blood samples were examined for HPV-specific CTLs or changes in levels of antibodies to HPV-16 or -18 E6 and E7 proteins and to vaccinia virus., Results: Vaccination with recombinant vaccinia was well tolerated in all patients with only mild to moderate local toxicity, and no serious adverse events were attributable to the vaccine. After a single vaccination, HPV-specific CTLs were found in four patients (HLA A1, A3, three patients; HLA A1, A24, one patient). Eight patients developed HPV-specific serological responses., Conclusions: This study confirmed the safety and immunogenicity of the vaccine in a proportion of those patients vaccinated. Additional clinical studies using TA-HPV in combination with an additional experimental vaccine for HPV-16 are currently under way.

Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers.

The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy.

The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.

Background: Oncofetal antigen 5T4 is defined by a monoclonal antibody raised against placental trophoblast membranes. Antigen 5T4 is reported to be expressed only on trophoblasts and neoplastic cells and would be a potentially useful marker for neoplastic cells., Materials and Methods: We evaluated the expression of 5T4 oncofetal antigen in gastric cancer by immunohistochemistry and investigated whether or not the expression of this antigen is correlated with the prognosis of patients with gastric cancer., Results: The 5T4 antigen was detected in 51.6% of primary gastric cancer (n = 62) and 5T4-reactivity was more frequently observed in carcinoma with lymph node metastasis (p < 0.05). The overall survival of 5T4-positive patients was significantly worse than that of 5T4-negative patients, analyzed by the log-rank test (p < 0.05), although multivariate analysis indicated that 5T4 expression was not an independent prognostic factor., Conclusion: The assessment of 5T4 expression may be useful in identifying gastric cancer patients with a poor prognosis.

It is now clear that many human tumour antigens can be recognised by the immune system. These tumour antigens can be classified into several groups including cancer-testis, differentiation, tissue specific, over-expressed, and viral-associated antigens. In many cases, there is a known molecular basis of carcinogenesis which provides the explanation for the differentiated expression of these antigens in tumours compared with normal cells. Improved understanding of the biology of the immune response, particularly of immune recognition and activation of T-cells, allow better design of vaccines. Pre-clinical comparative studies allow evaluation of optimal vaccine strategies which can then be delivered to the clinic. Currently, a range of cancer vaccines are being tested including those using tumour cells, proteins, peptides, viral vectors, DNA or dendritic cells. Ultimately, this research should give rise to an entirely new modality of cancer treatments.