Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines

Clinical trials are governed by an increasingly stringent regulatory framework, which applies to all levels of trial conduct. Study critical immunological endpoints, which define success or failure in early phase clinical immunological trials, require formal pre-trial validation. In this case study, we describe the assay validation process, during which the sensitivity, and precision of immunological endpoint assays were defined. The purpose was the evaluation of two multicentre phase I/II clinical trials from our unit in Southampton, UK, which assess the effects of DNA fusion vaccines on immune responses in HLA-A2+ patients with carcinoembryonic antigen (CEA)-expressing malignancies and prostate cancer. Validated immunomonitoring is being performed using ELISA and IFNγ ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNγ ELISPOT, was tested in a pre-trial study and found to be suitable for the detection of low frequency naturally occurring CEA- and prostate-derived tumour-antigen-specific T cells in patients with CEA-expressing malignancies and prostate cancer.

[1]  R. Young,et al.  Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1 , 1994, The Journal of experimental medicine.

[2]  D. Ankerst,et al.  Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT , 2005, BMC Immunology.

[3]  Holden T Maecker,et al.  Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides , 2008, BMC Immunology.

[4]  S. H. van der Burg,et al.  Induction of Tumor-Specific CD4+ and CD8+ T-Cell Immunity in Cervical Cancer Patients by a Human Papillomavirus Type 16 E6 and E7 Long Peptides Vaccine , 2008, Clinical Cancer Research.

[5]  J. Schlom,et al.  Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. , 1997, Cancer research.

[6]  F. Stevenson,et al.  Vaccination of human subjects expands both specific and bystander memory T cells but antibody production remains vaccine specific. , 2006, Blood.

[7]  M. Caulfield,et al.  Establishing Acceptance Criteria for Cell-Mediated-Immunity Assays Using Frozen Peripheral Blood Mononuclear Cells Stored under Optimal and Suboptimal Conditions , 2007, Clinical and Vaccine Immunology.

[8]  A. Iasonos,et al.  Safety and Immunogenicity Study of NY-ESO-1b Peptide and Montanide ISA-51 Vaccination of Patients with Epithelial Ovarian Cancer in High-Risk First Remission , 2008, Clinical Cancer Research.

[9]  P. Coulie,et al.  T-cell responses of vaccinated cancer patients. , 2003, Current opinion in immunology.

[10]  Xu Liu,et al.  Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus , 2001, Clinical Diagnostic Laboratory Immunology.

[11]  M. Carroll,et al.  Vaccination of colorectal cancer patients with TroVax given alongside chemotherapy (5-fluorouracil, leukovorin and irinotecan) is safe and induces potent immune responses , 2008, Cancer Immunology, Immunotherapy.

[12]  A. Ribas,et al.  Adenovirus MART-1–engineered Autologous Dendritic Cell Vaccine for Metastatic Melanoma , 2008, Journal of immunotherapy.

[13]  F. Marincola,et al.  Natural T cell immunity against cancer. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[14]  Krys J. Miller,et al.  Workshop on Bioanalytical Methods Validation for Macromolecules: Summary Report , 2001, Pharmaceutical Research.

[15]  B. Ludewig,et al.  Dendritic cell-based multi-epitope immunotherapy of hormone-refractory prostate carcinoma , 2006, Cancer Immunology, Immunotherapy.

[16]  C. Gouttefangeas,et al.  Toward the harmonization of immune monitoring in clinical trials: Quo vadis? , 2007, Cancer Immunology, Immunotherapy.

[17]  A. Sette,et al.  The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors. , 1998, Human immunology.

[18]  P. Moss,et al.  DNA fusion vaccines induce epitope-specific cytotoxic CD8(+) T cells against human leukemia-associated minor histocompatibility antigens. , 2006, Cancer research.

[19]  H. Sampson,et al.  Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study , 2006, BMC Immunology.

[20]  M. Atkins,et al.  Immunologic Monitoring of Cancer Vaccine Therapy: Results of a Workshop Sponsored by the Society for Biological Therapy , 2002, Journal of immunotherapy.

[21]  E. Thiel,et al.  Differences in T‐cell immunity toward tumor‐associated antigens in colorectal cancer and breast cancer patients , 2003, International journal of cancer.

[22]  J. Lathey.  Preliminary steps toward validating a clinical bioassay: A case study of the ELIspot assay , 2003 .

[23]  A. Eggermont,et al.  Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial. , 2000, Journal of immunological methods.

[24]  J. Schlom,et al.  Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. , 1995, Journal of the National Cancer Institute.

[25]  Sylvia Janetzki,et al.  A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. , 2002, Journal of immunological methods.

[26]  Herman Pieterse,et al.  Richtsnoer voor Good Clinical Practice (CPMP/ICH/135/95). , 2004 .

[27]  E. Celis,et al.  Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen. , 2002, Cancer research.

[28]  B. Boehm,et al.  CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. , 2003, Journal of immunological methods.

[29]  M. Roederer,et al.  T-cell quality in memory and protection: implications for vaccine design , 2008, Nature Reviews Immunology.

[30]  Sylvia Janetzki,et al.  Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) , 2007, Cancer Immunology, Immunotherapy.

[31]  A. Mackensen,et al.  The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays , 2007, Cancer Immunology, Immunotherapy.

[32]  D. Voehringer,et al.  Age-associated accumulation of CMV-specific CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1) , 2003, Experimental Gerontology.

[33]  Ashley M. Miller,et al.  Immune Monitoring in a Phase 1 Trial of a PSA DNA Vaccine in Patients with Hormone-Refractory Prostate Cancer , 2005, Journal of immunotherapy.

[34]  M. Baseler,et al.  A modified human ELISPOT assay to detect specific responses to primary tumor cell targets , 2004, Journal of Translational Medicine.

[35]  C. Herberts,et al.  A single naturally processed measles virus peptide fully dominates the HLA‐A*0201‐associated peptide display and is mutated at its anchor position in persistent viral strains , 2000, European journal of immunology.

[36]  C. Ottensmeier,et al.  DNA vaccines: precision tools for activating effective immunity against cancer , 2008, Nature Reviews Cancer.

[37]  J Cummings,et al.  Biomarker method validation in anticancer drug development , 2008, British journal of pharmacology.