Tools in Drug Formulation: Flow Cytometry and Data Analysis Protocols Janet Siebert CytoAnalytics

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Tools in Drug Formulation Flow Cytometry and
Data Analysis ProtocolsJanet
SiebertCytoAnalytics
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Overview

• Flow cytometry is a useful tool for measuring
  multiple parameters on cell-sized particles, e.g.
  peripheral blood cells
• Characteristics of both donor populations and
  individuals can be inspected using the right data
  analysis protocols
• Some of these characteristics may be appropriate
  biomarkers for disease targets, disease
  progression, or response to drug formulations

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Outline

• Introduction to CytoAnalytics
• 3 underlying concepts
• An experiment intracellular cytokine staining
  in breast cancer patients
• Take home message

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Introduction to CytoAnalytics

• CytoAnalytics helps researchers find and tell the
  stories in their data
• Outgrowth of collaboration with researchers in
  Karen Newells Institute for Bioenergetics at
  University of Colorado, Colorado Springs data
  from flow cytometry experiments
• Goal to provide fast, reliable,
  biologically-meaningful insight
• Analysts combine multiple data sources, apply
  appropriate algorithms from our library, return
  results to researchers in 1-3 business days
• Algorithms include visualization, statistical,
  and data mining techniques

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3 Underlying Concepts

• Flow cytometry
• Lyoplates
• Data normalization

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The Flow Cytometer

• Measures light and fluorescence from cell-sized
  particles in solution
• Ability to record multiple independent and
  quantitative measurements on a large number of
  cells
• Forward scatter, side scatter, and fluorescence
  (generally due to staining)

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Inside the Flow Cytometer
Forward scatter and side scatter
Hydrodynamic Focusing
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Graphic Displays of Resulting Data
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BD LyoplateTM Technology

• Plate-based assay containing lyophilized
  (freeze-dried) reagents
• Manufactured in controlled environment using
  robotics
• Mother plate used to create daughter plates
• Minimize of a source of variance in research
  results

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An ExperimentIntracellular Cytokine Staining
for Tumor Associated Antigens

• 41 healthy donors
• 21 with breast cancer
• Tumor-associated antigens (TAAs) HER2/neu, CEA,
  and MAGE-3 broadly expressed in breast cancer
  are candidates for T cell based immunotherapies
• Using intracellular cytokine flow cytometry,
  antigen-specific T cell responses to HER2/neu,
  CEA, and MAGE-3 measured in PBMCs (peripheral
  blood mononuclear cells)
• Responses also measured to CMV (eg pp65), SEB,
  HIV gag, and other peptide pools
• Levels of IFNg, IL-2 and TNFa measured on
  CD3/CD4 and CD3/CD4- T cells (Helper and
  Cytotoxic T cells)

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Use of Lyophilized 96 Well Plates
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Resulting Experimental Data 45,613 rows
(summarized)
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The Rich Analytical Environment

• Combine source data from instrument with sample
  and patient descriptive data

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Representative Data and Analysis
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Donor ProfilesCD4 IFNg, IL2, TNFa CD8 IFNg,
IL2, TNFa
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Overview of Normalization
For each donor, for each stimulus NormXi Xi
Xmin ------------ Xmax - Xmin
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A Normalization Example
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Normalized vs. Non-normalized Data
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Statistical Analysis with Normalized Data
CD4/IFNg TAAs lower than CMV plt.0103 CD8/IFNg
TAAs lower than CMV plt.004 CD8/IL-2 TAAs higher
than CMV plt.009
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The Research Team

• Margaret Inokuma1, Charles Schmitt3, Perry
  Haaland3, Douglas Petry1, Maria A. Suni1, Smita
  A. Ghanekar1, Daiva Gladding1, John F. Dunne1,
  Vernon C. Maino1, Mary L. Disis2, and Holden T.
  Maecker1
• 1BD Biosciences, San Jose, California
• 2University of Washington, Seattle, Washington
• 3BD Technologies, Research Triangle Park, North
  Carolina NC

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Underview

• Different donors have different patterns of
  reactions to different stimuli
• At a donor level, there are immunophenotypes
• We need to identify and monitor these phenotypes
  at various phases of the drug discovery and
  formulation process

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Flow Cytometry and Biomarkers

• Apoptotic CD34 cells are increased in patients
  with acute coronary syndrome and more extensive
  coronary artery disease
• Toll-like receptor 2 and 4 expression correlates
  with the extent and severity of coronary artery
  disease
• T lymphocytes in bronchoalveolar lavage (BAL) of
  patients with chronic obstructive pulmonary
  disease (COPD) produce more cytokines than in
  controls
• Multi-drug resistant multiple myeloma patients
  have significantly higher expression levels of
  adhesion molecules VLA-4 and ICAM-1 than do
  responders

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Biomarkers and Cellular AnalysisImproving Drug
Discovery and Formulation
Pre-clinical
Clinical Trials
Phase IV
IND
NDA
Phase I II
Phase III
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Take home messages

• 3 underlying concepts
• Flow cytometry
• Lyoplates
• Data normalization
• Big picture
• Disease heterogeneity and immunophenotypes
• Flow cytometry is a tool for collecting multiple
  parameters at a single cell level
• In RD, need to combine instrument readouts with
  clinical descriptive data and study in a rich
  analytical environment