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Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
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Featured Presentation FIRN Kona 2009
Featured Presentation FIRN Kona 2009
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Featured Presentation FIRN Kona 2009

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Feasibility of Monitoring Cell Mediated Immunity during Vaccine Trials

Feasibility of Monitoring Cell Mediated Immunity during Vaccine Trials

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  • 1. Feasibility of Monitoring Cell Mediated Immunity during Vaccine Trials FIRN – 2009 July 2009, Kona HI, USA Dr. Thomas O. Kleen Director, Business and Technology Development Cellular Technology Limited (C.T.L.) www.immunospot.com The views expressed are those of the author and do not necessarily correspond with the current views of CTL orSilversword agencies Haleakalā any regulatory (1998) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 1
  • 2. Immune Monitoring and Biomarker Screening during Vaccine Development is Good Science and Good Business • Accelerates time to market and save costs: • Potentially adds weeks, months or more to a product lifecycle under IP protection and facilitates the ability to enter the market ahead of a competing product • Go/no go decisions: • Enables to quickly refocus on alternative approaches if immune monitoring data indicate a lack of efficacy or should safety concerns arise during proof of concept, animal studies or any clinical phase of the development © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 2
  • 3. Objectives for Immune Monitoring and Biomarker Screening during Vaccine Development • Delivery of solid comparison data: • Immune monitoring and biomarker screening can be implemented during all pre-clinical and clinical phases of vaccine development • 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 ) 3
  • 4. Rationale for Monitoring Cell Mediated Immunity during Vaccine Trials • Cell Mediated Immunity (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, …..) • The emerging field of therapeutic vaccines has further highlighted the need to induce or modulate CMI in order to fight cancer, as well as autoimmune diseases and allergic responses © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 4
  • 5. 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 ) 5
  • 6. 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 ) 6
  • 7. 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 Accuracy, Precision, Specificity, Linearity and 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 ) 7
  • 8. 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 ) 8
  • 9. Widely Used Assay Technologies to Evaluate Cytokine Production and CMI • Enzyme-Linked Immunosorbent Assay (ELISA) Supernatant based • Luminex & Cytometric Bead Arrays (CBA) • Fluorescence-Activated Cell Sorter (FACS) • Intracytoplasmatic Cytokine Staining (ICS) • Tetramer staining Single Cell based • Pentamer staining • 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 ) 9
  • 10. Requirement for the Ability to Evaluate Multiple Functions, Molecules or Cytokines © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 10
  • 11. Advantage of Using Single Cell Cytokine Secretion by T-cells as Biomarker • Establishes 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/Th5/Th17/Thpp (CD4+ T-cells) and Tc1/Tc2 (CD8+ T-cells) based on cytokine signatures • Establishes the quantity (amount) of the secreted cytokine product in response to an antigen or vaccine • Establishes 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 potential adverse effects) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 11
  • 12. ELISPOT Assay’s Unique Qualification for Cytokine-Based Immune Monitoring 100.00 • Sensitivity: Routine detection limits of 1 in % IFN-γ positive cells FACS 10.00 500,000 events or better. Meaning in day to 1.00 0.10 day laboratory operations several to 0.01 101 102 103 104 105 106 0.8 hundred fold more sensitive than ELISA, 0.6 ELISA OD405 CBA, ICS, Tetramer, etc. 0.4 0.2 0.0 101 102 103 104 105 • Direct ex vivo cell frequencies: Measures # of IFN-γ Spots/Well 500 400 ELISPOT the physiological magnitude of T-cell 300 200 immunity, with no need for in vitro expansion 100 0 0 100 200 300 400 500 600 (not provided by ELISA, CBA, *ICSlow, Number of Cells *Tetramerlow, etc.) *(only applies to direct ex vivo low frequency T cell responses) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 12
  • 13. ELISPOT Assay’s Unique Qualification for Cytokine-Based Immune Monitoring (cont) • Samples not pharmacologically treated: No use of secretion inhibitors or cell permeable agents as with ICS and FACS • Robustness: Cell samples can be freeze thawed while maintaining highly reproducible results (with the right protocols) • Validation Capabilities: Assays can be validated under GLP with multiple cytokines and test systems (e.g., CTL Laboratories has validated the assay for its clients in human, mouse, monkey, and pig test systems) © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 13
  • 14. 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 cost-effectively tested per week (e.g., CTL tested 10,000 individual samples in less then 6 months) • Available 21 CFR Part 11 integration enabled equipment: For example, CTL’s ImmunoSpot® ELISPOT analysis equipment and software solutions are designed to be integrated into Part 11 compliant laboratory settings • Possibilities of standardization: Ranging for methods and reagents for clinical sample preservation to post trial final data read-outs the implementation of assay standardization is feasible © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 14
  • 15. Fresh versus frozen PBMC in ELISPOT Cytokine secretions are insensitive to a freeze IFN-g IL-2 thaw cycle: • ELISPOT assays with human PBMC measuring IFN-g (A), IL-2 (B), IL-4 (C), or IL-5 (D) IL-4 IL-5 • PBMC were tested either fresh (black circles) or thawed, 1 week after freezing (gray circles) Kreher et al., J. Immunol. Methods, (2003), 278:79-93 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 15
  • 16. The Choice of Test-system and Cytokines to monitor is Vital to Detect Relevant Responses in CD4 cells • Use of Adjuvants with CD4 cells: The choice of adjuvant during vaccination (i.e. IFA versus CFA) can switch peptide specific CD4+ recall responses form Th1 (IFN-g) to Th2 (IL-5) Yip et. Al,. J. Immunology, (1999) 162:3942-3949 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 16
  • 17. Use of Adjuvants with CD4 cells E.g. immunization with PLP 139-151 peptide plus CFA as adjuvant induces similar numbers of IFN-g and IL-2 producing CD4 cells, but IL-17 producing CD4 cells are induced by CFA only Tigno et al., J. Immunother, (2009), 32:389-398 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 17
  • 18. The Choice of Cytokines or Effector function to monitor is Vital to Detect Relevant Responses of CD8 Cells • Use of Adjuvants with CD8 cells : The choice of adjuvant during vaccination (i.e. CFA versus CPG) can switch peptide specific CD8+ recall responses between mainly DTH-mediating or mainly cytotoxic responses • Results in mice after immunization with class I restricted peptides in different type 1 adjuvants: IFN-g IL-2 IL-17 Killing DTH CFA +++ +++ +++ - +++ CpG + + - +++ - Tigno et al., J. Immunother, (2009), 32:389-398 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 18
  • 19. Use of Adjuvants with CD8 cells Immunization with “Uty” or SIINFEKL peptides plus CFA as adjuvant induce higher numbers of IFN-g and IL-2 producing CD8 cells than immunizations with CpG Tigno et al., J. Immunother, (2009), 32:389-398 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 19
  • 20. Use of Adjuvants with CD8 cells (cont) Immunization with “Uty” or SIINFEKL peptides plus CFA as adjuvant induce IL-17 producing CD8 cells while immunization with CpG does not SFU per 2 x 105 CD8 cells plated SFU per 1 x 106 splenocytes 150 120 IL - 17 p < 0.01 IL - 17 p < 0.05 125 100 100 * 80 75 60 * 50 40 25 20 0 * 0 * PBS Uty PBS Uty PBS SIINFEKL Uty Uty SIINFEKL Bulk response CD8 response Tigno et al., J. Immunother, (2009), 32:389-398 n=6 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 20
  • 21. Use of Adjuvants with CD8 cells (cont) Immunization with Uty or SIINFEKL peptides plus CpG as adjuvant induces stronger in vivo killing than immunization with CFA Tigno et al., J. Immunother, (2009), 32:389-398 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 21
  • 22. The Choice of Test-system and Cytokines or Molecule to monitor is Vital to Detect Relevant Responses • Example HIV: In humans HIV-peptide specific recall responses show a dissociated production of IFN-g and GzB (up to 40% of peptides only induce GzB) Patients recall to representative HIV peptides Kleen et al., AIDS, (2004), 18:383-392 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 22
  • 23. The Choice Cytokines or Secreted Molecules to monitor is Vital to Detect Relevant Responses CEF peptides do not induce direct ex vivo GzB or perforin in healthy human PBMC, but CD8 cells start secreting GzB and perforin 72 h after in vitro restimulation Ex vivo After 72h pre-activation Nowacki et al., Cellular Immunol, (2007), 247:36-48 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 23
  • 24. The Choice of Cytokines or Secreted Molecules to monitor is Vital to Detect Relevant Responses (cont) Dissociation of IFN-g vs. perforin and granzyme B allows distinction of memory vs. effector CD8 cells • Results in humans after recall with nominal peptide: IFN-g IL-2 GzB Perforin Memory +++ (+) - - Effector +++ (+) +++ +++ Nowacki et al., Cellular Immunol, (2007) 247:36-48 © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 24
  • 25. 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 ) 25
  • 26. Use of cryo-preserved reference sample PBMC • Existing Reference PBMC Library of 50 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 ulitisation of authentic, physilogical relevant cell activities without need for artifical or 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 ) 26
  • 27. Cryopreseved PBMC Reference Sample Show Low Intra- and Inter-Laboratory Variability of ELISPOT Results Intra-laboratory reproducibility of ELISPOT data. The three reference samples LP51, LP37 and LP58 were tested in three independent experiments with the same SOPs and materials by a single individual (A) or by three different individuals, whereby each of them thawed and processed the cells independently (B) In (C), for a separate experiment, the cells were thawed, processed and counted by a single individual and subsequently split into 3 identical aliquots for testing by 3 independent investigators. The mean and standard deviation for three replicate wells in each test is shown Data obtained in cooperation with Stéphanie McArdle during NEUCAPS multicenter trial © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 27
  • 28. 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 ) 28
  • 29. Serum Free Versus Serum Containing Test Media (cont) Serum Free Media FCS Containing Media Media CMVpp65 Media CMVpp65 PBMC sample 1 PBMC sample 2 Data obtained by Stéphanie McArdle during NEUCAPS multicenter trial © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 29
  • 30. Serum Free Versus Serum Containing Test Media (cont) Serum Free Media Prescreened Batch Tested human AB Serum Containing Media Media CMVpp65 Media CMVpp65 PBMC sample 1 PBMC sample 2 Data obtained by Stéphanie McArdle during NEUCAPS multicenter trial © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 30
  • 31. ELISPOT as Validated Cellular Readout of a Functional Assay 96 well mircotiter plate coated with A detection antibody Secreting cell – e.g., antigen- B stimulated T cell secreting cytokine Plate-bound secretory product C (cytokine) after cells are washed away Product-specific detection antibody D with linked enzyme Enzyme catalyzed chromogen E spot 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 ) 31
  • 32. 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 ) 32
  • 33. Acknowledgements Dr. Paul V. Lehmann Dr. Magdalena Mary-Lehmann Dr. Oleg Targoni Dr. Wenji Zhang Dr. Stéphanie McArdle & All 11 participating Laboratories during the NEUCAPS – I multicenter trials Dr. Stefanie Kuerten Dr. Justin Tingo © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 33
  • 34. Thank You! Questions? © Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 34

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