Recent Submissions

  • Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation.

    Elkord, Eyad; Clinical Immunotherapy Laboratory, Department of Medical Oncology, University of Manchester, Wilmslow Road, Manchester M204BX, UK. eelkord@picr.man.ac.uk (2009-08-15)
    Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.
  • Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4).

    Elkord, Eyad; Dangoor, Adam; Burt, Deborah J; Southgate, Thomas D; Daayana, Sai; Harrop, Richard; Drijfhout, Jan W; Sherlock, David J; Hawkins, Robert E; Stern, Peter L; CR UK Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK, eelkord@picr.man.ac.uk. (2009-02-17)
    We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.
  • Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.

    Karanam, Balasubramanyam; Gambhira, Ratish; Peng, Shiwen; Jagu, Subhashini; Kim, Dae-Jin; Ketner, Gary W; Stern, Peter L; Adams, Robert J; Roden, Richard B S; Department of Pathology, The Johns Hopkins University, Baltimore, MD 21231, USA. (2009-02-11)
    A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.
  • Recommendations for cervical cancer prevention in Asia Pacific.

    Garland, Suzanne M; Cuzick, Jack; Domingo, Efren J; Goldie, Sue J; Kim, Young-Tak; Konno, Ryo; Parkin, D Maxwell; Qiao, You-Lin; Sankaranarayanan, Rengaswamy; Stern, Peter L; Tay, Sun Kuie; Bosch, F Xavier; Department of Microbiology and Infectious Diseases, The Royal Women's Hospital, Melbourne, Victoria, Australia. Sue.Garland@the womens.org.au (2008-08-19)
    Asia Oceania includes countries from both the Asia Pacific region and Australasia, which cover very diverse geographical areas and populations as well as bearing 52% of the cervical cancer burden in the world. Human papillomavirus (HPV) genotype distribution in women with normal cytology varies between countries in this region, as well as with the distribution typically observed in worldwide estimates or in Western countries. HPV-16 remains the predominant oncogenic type for high-grade cervical dysplasia and cervical cancer across the region, and HPV-18 is generally among the five most common types. HPV-58 is commonly found in cervical cancer as well as in women with normal cytology, and HPV-31, 33 and 35 are relatively less frequent in these regions compared to the West. While screening programmes have been proposed and implemented in several populations, successful programmes are rather limited and the majority of countries still have no or minimal screening services. Prophylactic HPV vaccination will only be feasible when it becomes affordable, thus the current priority and the short-term goal for cervical cancer control is to identify feasible and effective screening measures, and to find the most effective way to combine vaccination with sustainable screening programmes. This Regional Report has carefully described the disease burden of HPV and cervical cancer and the current situations in cervical cancer prevention for many countries in the Asia Oceania region. These data identify the many challenges and opportunities to be considered for policy decisions for cervical cancer control. Furthermore, this report presents the results of advanced decision analytic models calibrated to countries in the region that provide early insight into what strategies are most promising and those likely to be cost-effective and affordable. It thus provides a synthesis of the available evidence-based scientific information, in the context of a significant and systematic international review, that is likely to be useful to governments and public health providers.
  • An MVA-based vaccine targeting the oncofetal antigen 5T4 in patients undergoing surgical resection of colorectal cancer liver metastases.

    Elkord, Eyad; Dangoor, Adam; Drury, Noel L; Harrop, Richard; Burt, Deborah J; Drijfhout, Jan W; Hamer, Caroline; Andrews, Danielle; Naylor, Stuart; Sherlock, David J; Hawkins, Robert E; Stern, Peter L; CRUK Immunology, Medical Oncology Groups, Paterson Institute for Cancer Research, University of Manchester, Christie Hospital NHS Trust, Manchester, UK. (2009-03-17)
    We investigated the use of a therapeutic vaccine, TroVax in patients undergoing surgical resection of colorectal cancer liver metastases. Systemic immunity generated by vaccination before and after resection of metastases was measured in addition to assessing safety and analyzing the function and phenotype of tumor-associated lymphocytes. Twenty patients were scheduled to receive 2 TroVax vaccinations at 2-week intervals preoperatively and 2 postoperatively; if immune responses were detected, 2 further vaccinations were offered. Blood was taken at trial entry and 2 weeks after each vaccination; tumor biopsies were collected at surgery. 5T4-specific cellular responses were assessed by lymphocyte proliferation and enzyme-linked immunosorbent spot, with antibody responses by enzyme-linked immunosorbent assay. Immunohistochemistry characterized the phenotype of tumor-infiltrating lymphocytes. Seventeen of 19 colorectal cancer patients showed 5T4 expression in the liver metastases or surrounding stroma and 18 mounted a 5T4-specific cellular and/or humoral response. In patients who received at least 4 vaccinations and potentially curative surgery (n=15), those with above median 5T4-specific proliferative responses or T-cell infiltration into the resected tumor showed significantly longer survival compared with those with below median responses. Seven of 8 patients who had preexisting proliferative responses to 5T4 were longer-term survivors; these patients showed significantly higher proliferative responses after vaccination than those who subsequently died. These data suggest that the magnitude of 5T4 proliferative responses and the density of CD3 cells in colorectal cancer liver metastases are associated with longer survival. These observations warrant more studies to identify the precise underlying mechanisms.
  • CD4+ T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25+ T cells.

    Elkord, Eyad; Burt, Deborah J; Drijfhout, Jan W; Hawkins, Robert E; Stern, Peter L; Department of Immunology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. eelkord@picr.man.ac.uk (2008-06)
    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.
  • Novel therapeutic strategies by regulatory T cells in allergy.

    Elkord, Eyad; Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. eelkord@picr.man.ac.uk (2008)
    Natural CD4+CD25+FoxP3+ regulatory T cells (Treg) actively suppress physiological and pathological responses, therefore playing a critical role in controlling peripheral tolerance to self antigens and maintaining immune homeostasis. In normal individuals, natural Treg and interleukin- 10-secreting Treg are able to suppress Th2 responses to allergens, whereas lower levels of Treg or defect in their functionality have been described as potential mechanisms for inducing allergic diseases. In animal models, adoptive transfer of CD4+CD25+Treg has been shown as a promising strategy for preventing or treating allergic disorders. Recent studies show that induction of Treg activity is associated with suppression of allergic responses in allergic patients treated with specific immunotherapy. Herein, I review the potential of Treg as exciting targets for developing new immunotherapeutic strategies for treating allergic diseases.
  • Adoptive transfer of T(reg) depleted autologous T cells in advanced renal cell carcinoma.

    Thistlethwaite, Fiona C; Elkord, Eyad; Griffiths, Richard W; Burt, Deborah J; Shablak, Alaaeldin; Campbell, John D M; Gilham, David E; Austin, Eric B; Stern, Peter L; Hawkins, Robert E; Cancer Research UK Department of Medical Oncology, University of Manchester and Christie Hospital NHS Foundation Trust, Manchester, UK. fthistlethwaite@PICR.man.ac.uk (2008-05)
    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.