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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

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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


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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)


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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

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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


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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


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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.

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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


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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)

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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

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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)


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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)


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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)

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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)


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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

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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

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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)

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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

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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


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Validated Computer Assisted Image Acquisition,
Counting and Analysis of Data


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Thank You!

Questions?


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Invited Presentation at the World Vaccine Congress Lyon 2008

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