• Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

      Brooks, Suzanne E.; Bonney, Stephanie A.; Lee, Cindy; Publicover, Amy; Khan, Ghazala; Smits, Evelien L.J.; Sigurdardottir, Dagmar; Arno, Matthew; Li, Demin; Mills, Ken I.; et al. (Public Library of Science, 2015-10-22)
      Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.
    • Analogue peptides for the immunotherapy of human acute myeloid leukemia

      Hofmann, Susanne; Mead, Andrew; Malinovskis, Aleksandrs; Hardwick, Nicola R.; Guinn, Barbara-Ann; University of Bedfordshire (Springer, 2015-10-05)
      The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies.
    • Infrequent expression of the cancer-testis antigen, PASD1, in ovarian cancer

      Khan, Ghazala; Brooks, Suzanne E.; Mills, Ken I.; Guinn, Barbara-Ann; University of Bedfordshire; Southampton General Hospital; Queen’s University Belfast (Libertas Academica, 2015-08)
      Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.
    • Is there a need to identify novel tumour antigens as targets for immunotherapy clinical trials for the removal of minimal residual disease in haematological malignancies?

      Guinn, Barbara-Ann; University of Bedfordshire (ACT Publishing Group, 2015-07)
      Despite the identification of many tumour antigens with the potential to act as targets for cancer vaccines and/or T-cell therapies very few have been used in clinical trials to date. This led to the timely development of a criteria which identified the ideal characteristics of tumour antigens which should be actively pursued for use in immunotherapy clinical trials. A list of 75 antigens were assessed against these criteria and although none harboured all of the characteristics identified as desirable, a number did show many of the characteristics identifying them as worthy of further pursuit to enable an organised development towards immunotherapy clinical trials. The study highlighted the benefit of focussing on a short list of antigens which would enable the rapid progress of a smaller number of antigens into clinical trials as targets for immunotherapy. However the antigens expressed by solid tumours often differ to those expressed by haematological malignancies, leading to this editorial which states the need for a similar study prioritising tumour antigens for use in clinical trials of haematological malignancies, independently of solid tumours. We also debate the importance of looking for new antigens in cancers in which few targets are known and discuss the importance of tumour antigens as biomarkers of disease diagnosis, stage and survival.
    • A novel zinc finger gene, ZNF465, is inappropriately expressed in presentation acute myeloid leukaemia cells

      Collin, Joseph F.; Wells, James W.; Czepulkowski, Barbara; Lyne, Linden; Duriez, Patrick J.; Banham, Alison H.; Mufti, Ghulam J.; Guinn, Barbara-Ann; King's College London; University of Oxford; et al. (Wiley, 2015-05)
      To increase our knowledge of leukaemia-associated antigens, especially in acute myeloid leukaemia (AML) M4, we prepared a phage display cDNA library using mRNA from the bone marrow cells of a patient with AML M4 at diagnosis. We immunoscreened 10(6) pfu with autologous sera and identified an antigen which we named GKT-AML8. The cDNA showed more than 99% similarity to a sequence on 2q21.2 and 95% sequence similarity to a sequence on 19q13.3. These genes were named ZNF465 and ZNF466, respectively, following HUGO Gene Nomenclature Committee (HGNC) guidelines. Expressed sequence tag data suggests that both genes are transcriptionally active. ZNF465 and ZNF466 encode a 5' krüppel associated box domain typical of negative regulators of gene transcription. We have confirmed the translational start site in the +1 frame in a near-Kozak sequence that produces a 102 amino acid polypeptide from ZNF465. The high level of sequence similarity between ZNF465 and ZNF466 makes their transcripts almost indistinguishable by real-time polymerase chain reaction (RT-PCR). However, GKT-AML8 showed most sequence similarity to ZNF465 and no transcript matching the 3' ZNF466 sequence could be detected in patient samples or healthy volunteers. ZNF465/466 expression was detectable in 12/13 AML and 10/14 chronic myeloid leukaemia patients' samples but not in normal donor peripheral blood (0/8) or 0/3 bone marrow samples which had been separated into CD34(+) and CD34(-) samples. The altered expression of ZNF465/466 in patients' samples and its absence in healthy donor haematopoietic samples indicate that ZNF465 is overexpressed in early myeloid disease and as such may represent a promising target for immunotherapy.
    • The future of publishing scientific data: is it time to accept the wider publication of null data?

      Guinn, Barbara-Ann; University of Bedfordshire (E Cronicon, 2014-12)
      One of the most self-limiting dogmas’ which scientists submit themselves to is the avoidance of publishing negative data. This reflected the fact that authors generally do not write up and submit null data. The avoidance of publishing negative data (by authors and journals) ensures we only show experiments that worked and represent a clear story of success. Experiments which the research community remains ignorant of and which thus be repeated wasting the limited money currently available for research from the government and charities, and time which could be spent more productively, moving the field forward more rapidly. But how should the science community publish negative data? This Editorial discusses some of the concerns regarding publishing null data and ways we can move the bioscience forward through it's publication.
    • 8th international conference on oncolytic virus therapeutics

      Guinn, Barbara-Ann; Braidwood, Lynne; Parker, Alan; Peng, Kah-Whye; Seymour, Leonard (Mary Ann Liebert, 2014-12)
      The 8th International Conference on Oncolytic Virus Therapeutics meeting was held from April 10-13, 2014, in Oxford, United Kingdom. It brought together experts in the field of oncolytics from Europe, Asia, Australasia, and the Americas and provided a unique opportunity to hear the latest research findings in oncolytic virotherapy. Presentations of recent work were delivered in an informal and intimate setting afforded by a small group of attendees and an exquisitely focused conference topic. Here we describe the oral presentations and enable the reader to share in the benefits of bringing together experts to share their findings.
    • Oral tolerance to cancer can be abrogated by T regulatory cell inhibition

      Whelan, Maria C.; Casey, Garrett; Larkin, John O.; Guinn, Barbara-Ann; O'Sullivan, Gerald C.; Tangney, Mark; University College Cork; University of Bedfordshire (2014-05-15)
      Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.
    • Vaccination against myeloid leukaemias using newly defined antigens

      Hofmann, Susanne; Khan, Ghazala; Boncheva, Viktoriya Bogdanova; Greiner, Jochen; Guinn, Barbara-Ann (Oxford University Press, 2014)
      First complete remission rates are high in patients with acute myeloid leukaemia (AML), with some variation depending on the presence of specific cytogenetic and molecular aberrations. However, the remission is often not long lasting and relapse occurs after standard chemotherapy within two years. Besides chemotherapy, non-specific immunotherapy in the form of allogeneic haematopoietic stem cell transplantation (HSCT) is an integral part of consolidation and salvage therapy in the treatment of AML. A large number of leukaemia-associated antigens (LAAs) that can act as potential targets for specific immunotherapy have been identified, and the number is still increasing. To date, several of these antigens are being utilized in clinical vaccination trials, either as active specific immunotherapy in form of peptide vaccination or as passive specific immunotherapy as adoptive cell therapies. This chapter reviews the role of newly defined LAAs as well as the results of already performed clinical vaccination trials with known LAAs.
    • PASD1: a promising target for the immunotherapy of haematological malignancies

      Khan, Ghazala; Denniss, Frances; Mills, Ken I.; Pulford, Karen; Guinn, Barbara-Ann; University of Bedfordshire (OMICS International, 2013-10)
      In general, there is a lack of good immunotherapy targets within the spectrum of haematological malignancies. However haematopoietic stem cell transplants and continuing antigen discovery have allowed further insight into how further improvements in outcomes for patients might be achieved. Most patients with haematological malignancies can be treated with conventional therapies such as radio- and chemotherapy and will attain first remission. However the removal of residual diseased cells is essential to prevent relapse and its associated high mortality. PASD1 is one of the most tissue restricted cancer-testis (CT) antigens with expression limited to primary spermatagonia in healthy tissue. However, characterisation of PASD1 expression in cancers has been predominantly focussed on haematological malignancies where the inappropriate expression of PASD1 was first identified. PASD1 has one of the highest frequencies of expression of all CT antigens in acute myeloid leukaemia, with some suggestion of its role as a biomarker in diffuse large B-cell lymphoma. Here we describe the characterisation of the function and expression patterns of PASD1 in cell lines and primary tissues. Development of DNA vaccines targeting PASD1 epitopes demonstrate effective ex vivo T-cell responses in terms of IFNγ secretion and tumour cell killing. Of particular note these vaccines have led to the destruction of cells which process and present endogenous PASD1 indicating that effectively primed CTLs could kill PASD1-positive tumour cells.
    • High and low, but not intermediate, PRAME expression levels are poor prognostic markers in myelodysplastic syndrome at disease presentation

      Liberante, Fabio G.; Pellagatti, Andrea; Boncheva, Viktoriya Bogdanova; Bowen, David T.; Mills, Ken I.; Boultwood, Jacqueline; Guinn, Barbara-Ann; University of Bedfordshire; Leeds Teaching Hospitals NHS Trust; Queens University Belfast; et al. (Wiley, 2013-07)
    • Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6

      Witzens-Harig, Mathias; Hose, Dirk; Junger, Simone; Pfirschke, Christina; Khandelwal, Nisit; Umansky, Ludmilla; Seckinger, Anja; Conrad, Heinke; Brackertz, Bettina; Reme, Thierry; et al. (American Society of Hematology, 2013-04-19)
      Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.
    • New targets for the immunotherapy of colon cancer—does reactive disease hold the answer?

      Boncheva, Viktoriya Bogdanova; Bonney, S.A.; Brooks, Suzanne E.; Tangney, Mark; O'Sullivan, Gerald C.; Mirnezami, A.; Guinn, Barbara-Ann (Nature Publishing Group, 2013-03-15)
      Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women, posing a serious demographic and economic burden worldwide. In the United Kingdom, CRC affects 1 in every 20 people and it is often detected once well established and after it has spread beyond the bowel (Stage IIA-C and Stage IIIA-C). A diagnosis at such advanced stages is associated with poor treatment response and survival. However, studies have identified two sub-groups of post-treatment CRC patients--those with good outcome (reactive disease) and those with poor outcome (non-reactive disease). We aim to review the state-of-the-art for CRC with respect to the expression of cancer-testis antigens (CTAs) and their identification, evaluation and correlation with disease progression, treatment response and survival. We will also discuss the relationship between CTA expression and regulatory T-cell (Treg) activity to tumorigenesis and tumor immune evasion in CRC and how this could account for the clinical presentation of CRC. Understanding the molecular basis of reactive CRC may help us identify more potent novel immunotherapeutic targets to aid the effective treatment of this disease. In this review, based on our presentation at the 2012 International Society for the Cell and Gene Therapy of Cancer annual meeting, we will summarize some of the most current advances in CTA and CRC research and their influence on the development of novel immunotherapeutic approaches for this common and at times difficult to treat disease.
    • The innovative evolution of cancer gene and cellular therapies

      Lam, P.; Khan, Ghazala; Stripecke, R.; Hui, K.M.; Kasahara, N.; Peng, Kah-Whye; Guinn, Barbara-Ann (Nature Publishing Group, 2013-02-01)
      We provide an overview of the latest developments in cancer gene therapy--from the bench to early-stage clinical trials. We describe the most recent work of worldwide teams including experienced scientists and clinicians, reflecting the recent emergence of gene therapy from the 'Valley of Death'. The treatment efficacy of clinical gene therapy has now been shown in a number of diseases including cancer and we are observing a renewed interest by big pharmaceutical and biotechnology companies most obviously demonstrated by Amgen's acquisition of Biovex for up to USD$1 billion. There is an opportunity to be cautiously hopeful regarding the future of gene therapy in the clinic and we review here some of the most recent progress in the field.
    • Predicting ecological regime shift under climate change: new modelling techniques and potential of molecular-based approaches

      Stafford, Richard; Smith, V.Anne; Husmeier, Dirk; Grima, Thomas; Guinn, Barbara-Ann; Bournemouth University; University of St Andrews; University of Glasgow; University of Bedfordshire (Current Zoology, 2013)
      Ecological regime shift is the rapid transition from one stable community structure to another, often ecologically inferior, stable community. Such regime shifts are especially common in shallow marine communities, such as the transition of kelp forests to algal turfs that harbour far lower biodiversity. Stable regimes in communities are a result of balanced interactions between species, and predicting new regimes therefore requires an evaluation of new species interactions, as well as the resilience of the ‘stable’ position. While computational optimisation techniques can predict new potential regimes, predicting the most likely community state of the various options produced is currently educated guess work. In this study we integrate a stable regime optimisation approach with a Bayesian network used to infer prior knowledge of the likely stress of climate change (or, in practice, any other disturbance) on each component species of a representative rocky shore community model. Combining the results, by calculating the product of the match between resilient computational predictions and the posterior probabilities of the Bayesian network, gives a refined set of model predictors, and demonstrates the use of the process in determining community changes, as might occur through processes such as climate change. To inform Bayesian priors, we conduct a review of molecular approaches applied to the analysis of the transcriptome of rocky shore organisms, and show how such an approach could be linked to measureable stress variables in the field. Hence species-specific microarrays could be designed as biomarkers of in situ stress, and used to inform predictive modelling approaches such as those described here.
    • An analogue peptide from the cancer testis antigen, PASD1, induces CD8+ T cell-responses against naturally processed peptide.

      Hardwick, Nicola R.; Buchan, Sarah; Ingram, Wendy; Khan, Ghazala; Vittes, Gisella; Rice, Jason; Pulford, Karen; Mufti, Ghulam J.; Stevenson, Freda; Guinn, Barbara-Ann; et al. (Cancer Research Institute, 2013)
      We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.
    • Identification and validation of targets for cancer immunotherapy: from the bench-to-bedside

      Khan, Ghazala; Brooks, Suzanne E.; Denniss, Frances; Sigurdardottir, Dagmar; Guinn, Barbara-Ann; University of Bedfordshire; University of Southampton; King’s College London; University of Tübingen (InTech OpenAccess, 2013)
    • PASD1 (PAS domain containing 1)

      Khan, Ghazala; Guinn, Barbara-Ann (2013)
    • SSX2IP Synovial Sarcoma, X breakpoint 2 interacting protein

      Khan, Ghazala; Guinn, Barbara-Ann; University of Bedfordshire (Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2012-03)
    • Expression of CD66 in non-Hodgkin lymphomas and multiple myeloma.

      Guinn, Barbara-Ann; Bosslet, Klaus; Lee, Cindy; Richardson, Deborah; Orchard, Kim H.; University of Bedfordshire (Wiley, 2011-12)