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Invited Presentation at the World Vaccine Congress Lyon 2008

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Assessing Immunogenicity and Safety by High Resolution Monitoring of Cell Mediated Immunity

Assessing Immunogenicity and Safety by High Resolution Monitoring of Cell Mediated Immunity

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    Invited Presentation at the World Vaccine Congress Lyon 2008 Invited Presentation at the World Vaccine Congress Lyon 2008 Presentation Transcript

    • by: Assessing Immunogenicity and Safety by High Resolution Monitoring of Cell Mediated Immunity World Vaccine Congress 2008 CLINICAL TRIALS AND TRIBULATIONS 08 October 2008, Palais des Congrès de Lyon, France Dr. Thomas O. Kleen Director, Business and Technology Development Cellular Technology Limited (C.T.L.) www.immunospot.com Homage to Mondrian (1872 -1944) The views expressed are those of the author and do not necessarily correspond with the current views of CTL or any regulatory agencies © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 1
    • by: Presentation Outline • Objectives for Immune Monitoring and Biomarker Screening • Rationale for Monitoring Cell Mediated Immunity (CMI) • Current Challenges facing the Broad Implementation of Monitoring CMI • Required and Desirable Characteristics of Assays for Monitoring CMI in regulated Environments • Assays used to monitor CMI and Cytokines as Biomarkers • ELISPOT Assays Unique Qualification for Cytokine-Based Immune Monitoring and Standardization Strategies © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 2
    • by: Company Profile: Cellular Technology Limited (C.T.L.) • Founded in 1999 • Headquarters and laboratories located in Shaker Heights, Ohio, USA • Sales and marketing subsidiaries in Germany and China • Core business areas: • Contract Research Services (CTL Laboratories LLC) • Image Analysis Equipment (CTL Analyzers LLC) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 3
    • by: Objectives for Immune Monitoring and Biomarker Screening during Vaccine Development • Accelerate time to market and save costs: • Potential to add weeks, months or more to a product lifecycle under IP protection and/or can allow to enter the market ahead of a competing product • Go/no go decisions: • Quickly refocus on alternative approaches if immune monitoring data indicate a lack efficacy and/or should safety concerns arise during proof of concept, animal studies or any clinical phases of development © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 4
    • by: Objectives for Immune Monitoring and Biomarker Screening during Vaccine Development (cont) • Delivery of solid comparison data: • Immune monitoring and biomarker screening can be implemented during all pre-clinical and clinical phases of vaccine development • To increase chances of a successful clinical trial: • High resolution, GLP compliant immune monitoring provides regulatory acceptable data © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 5
    • by: Rationale for Monitoring Cell Mediated Immunity during Vaccine Trials • CMI is a critical component during most immunological responses; involved in infectious diseases, cancer, and autoimmunity • Basic research into correlates of protection has exposed the limitations of vaccine approaches that rely solely on antibody responses to confer protection against pathogens (e.g., HIV, HCV, TB, Smallpox, and more) • The emerging field of therapeutic vaccines has further highlighted the need to induce CMI in order to fight cancer, as well as atopic or autoimmune diseases © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 6
    • by: Current Challenges facing Broad Implementation of CMI Monitoring • Monitoring technologies with sufficient high resolution are often perceived to be too costly, complex, and time consuming to be routinely utilized with hundreds or even thousands of samples. This applies in particular to technologies that aim to detect crucial low frequency T-cell responses. • Common misconceptions surrounding regulatory acceptance of CMI and biomarker data have to be overcome. © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 7
    • by: Current Challenges facing Broad Implementation of CMI Monitoring (cont) • Limited awareness exists in certain segments of the industry regarding recent advances in: • Cell sample cryo-preservation and thawing procedures • Cost effective, sensitive, single cell-based, high throughput capable assay technologies • Validation procedures for cell-based assay systems • Standardization reagents and procedures for cell-based assay and test systems © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 8
    • by: Required Characteristics of Assays to Monitor CMI in Regulated Environments • Measures a physiological and clinically relevant response • Is a reproducible, reliable assay for testing serial samples • Lends itself to validation (possibility of determining, e.g., Accuracy, Precision, Specificity, Linearity, Limits of Detection) • Has a meaningful sensitivity that is able to detect low frequency cells (Memory T-cells are frequently rare as 1 in 100,000 or less) • Available data analysis equipment must be able to be integrated in 21 CFR Part 11 compliant laboratory settings (i.e., system validation, computer generated audit trails) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 9
    • by: Desirable Features of Assays for Monitoring CMI in Regulated Environments • Performs identical with fresh and previously frozen samples • Can be standardized; to enable, for example, inter-study comparisons to make data more robust for multicenter or large clinical trials • Uses the least amount of cells and clinical sample material • Capability to be run in high throughput mode to accommodate large-volume testing with hundreds of samples a day • Availability of automated, high throughput capable data read-out equipment including data analysis software to insure objectivity © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 10
    • by: Commonly Available Assay Technologies to Evaluate Cytokine Production and CMI • Enzyme-Linked Immunosorbent Assay (ELISA) Supernatant based • Cytometric Bead Array (CBA) • Fluorescence-Activated Cell Sorter (FACS) • Intracytoplasmatic Cytokine Staining (ICS) • Tetramer staining • Pentamer staining Single Cell based • Surface marker staining • Enzyme-Linked Immunospot Assay (ELISPOT) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 11
    • by: Advantage of Using Single Cell Cytokine Secretion by T-cells as Biomarker • Establish the quality of an immune response, i.e., the type of products T-cells secrete in response to a vaccine candidate, allowing the differentiation between Th1/Th2/Th17 (CD4+ T-cells) and Tc1/Tc2 (CD8+ T-cells) based on cytokine signatures • Establish the quantity (amount) of the secreted cytokine product in response to an antigen or vaccine • Establish the frequency (clonal sizes) of the responding cells to an antigen or vaccine, giving an indication about scale or potency of the immune response (or adverse effects) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 12
    • by: ELISPOT Assay’s Unique Qualification for Cytokine-Based Immune Monitoring • Sensitivity: Routine detection limits of 1 in 100.00 % IFN-γ positive cells FACS 10.00 500,000, or better. That is several ten to 1.00 0.10 hundred folds more sensitive than ELISA, CBA, 0.01 ICS, Tetramer, etc. 0.8 101 102 103 104 105 106 ELISA 0.6 OD405 • Direct ex vivo cell frequencies: Measures the 0.4 0.2 0.0 physiologic magnitude of T-cell immunity, with 101 102 103 104 105 # of IFN-γ Spots/Well no need for in vitro expansion (not provided by 500 400 ELISPOT ELISA, CBA, *ICSlow, *Tetramerlow, etc.) 300 200 100 • Samples not pharmacologically treated: No use 0 0 100 200 300 400 500 600 Number of Cells of secretion inhibitors or cell permeable agents as with ICS and FACS *(only applies to low frequency T cell responses) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 13
    • by: ELISPOT Assay’s Unique Qualification for Cytokine-Based Immune Monitoring (cont) • Robustness: Cell samples can be IFN-g IL-2 freeze thawed while maintaining highly reproducible results • Validation Capabilities: Assays IL-4 IL-5 can be validated under GLP with multiple cytokines and test systems (e.g., CTL Laboratories has validated for its clients Performance Fresh versus frozen PBMC in human, mouse, monkey, and pig J. Immunol. Methods, 2003, 278 :79– 93 test systems, among others) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 14
    • by: ELISPOT Assay’s Unique Qualification for Cytokine-Based Immune Monitoring (cont.) • Scalable high throughput capability: In trained and appropriately setup up laboratory environments, up to 450 clinical samples can be tested per week (e.g., CTL Laboratories tested 10,000 individual samples in less then 6 months) • 21 CFR Part 11 integration enabled equipment: For example, CTL Analyzer’s line of ImmunoSpot® ELISPOT analysis equipment and software solutions, which are designed to be integrated into Part 11 compliant laboratory settings • Possibilities of standardization: Starting from methods and reagents for clinical sample preservation to final data read-outs, implementation of assay standardization is feasible © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 15
    • by: Standardization Strategies for CMI monitoring and ELISPOT • Harmonize sample collection at clinical sites • Training and qualification of all clinical sites in proper sample handling, PBMC processing, cryopreservation and shipping standard operating procedures (SOPs) • Harmonize methods across all participating laboratories • Implementation of detailed SOPs for cell thawing, cell counting, assay procedures and the analysis of assay results • Standardize all assay materials, including plates, antibodies, media and enzymes (always re-qualify new lots!) • Utilize cell-based reference sample PBMC to optimize assays and compare performance between laboratories © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 16
    • by: Advantages of serum-free cryopreservation reagents and assay media • Use of standardized serum-free cryopreservation reagents for PBMC yields a <95% post-thaw viability, maximizing clinical sample utilization (in combination with optimized freeze-thaw procedures) • Serum-containing undefined products like Fetal Bovine Serum (FBS), Fetal Calf Serum (FCS), as well as human ABO serum in cryo-preservation and assay media can cause significant assay background noise and artifacts rooted in large intra-batch and performance variations • Utilization of bovine products is prohibitive for all clinical trials involving international shipping of samples, based on strict import restrictions of many countries due to BSE concerns (mad cow disease) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 17
    • by: Use of cryo-preserved reference sample PBMC • Reference PBMC library of 40 healthy, HLA-typed and immune- characterized individuals with up to 10 billion cells per individual • Each sample has an established cytokine secretory reactivity to 32 individual T cell peptide epitopes of Cytomegalo-, Epstein-Barr, and Flu virus (CEF), as well as to 5 proteins from Candida, Dust Mite, Mumps, Tetanus, and PPD • Used for assay validation and protocol development through authentic, physilogical relevant cell activities without need for artifical, artifact prone mitogenic stimulation (PHA. ConA) • Enables realistic benchmarking of laboratory perfomance and internal controls during clinical trials without scarifcing precious clinical samples © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 18
    • by: ELISPOT as Validated Cellular Readout of a Functional Assay 96 well mircotiter plate coated with A detection antibody Secreting cell – e.g., antigen-stimulated B T cell secreting cytokine Plate-bound secretory product C (cytokine) after cells are washed away Product-specific detection antibody with D linked enzyme Enzyme catalyzed chromogen spot E development or fluorescence label PVDF-membrane Antigen-stimulated cell Enzyme-coupled detection antibody Secreted cytokine Capture antibody Chromogen/Fluorescence label © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 19
    • by: Validated Computer Assisted Image Acquisition, Counting and Analysis of Data © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 20
    • by: Thank You! Questions? © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 21