Potency Testing for an Autologous Cellular Immunotherapy

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Potency Testing for an Autologous Cellular
Immunotherapy

• Nicole Provost, PhD
• VP Product Development
• Dendreon Corporation
• February 9, 2006

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Overview

• Introduction to the process and product
• Model system healthy donor apheresis cells
• Molecular tools and cellular assays
• Correlating antigen presentation activity with
  cell phenotype
• Justifying potency assays
• Tracking potency over time
• Comparing potency data with clinical outcomes
• Q A

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Sipuleucel-T (Provenge) Manufacturing Process
Day 1 Leukapheresis
Day 2-3 Sipuleucel-T is manufactured
Day 3-4 Patient is infused
Apheresis Center
Dendreon
Doctors Office
COMPLETE COURSE OF THERAPY 3 CYCLES
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Cellular Immunotherapy with Sipuleucel-T
APC takes up the antigen
Recombinant Prostatic Acid Phosphatase (PAP)
antigen combines with resting antigen presenting
cell (APC)
Fully activated, the APC is now sipuleucel-T
Antigen is processed and presented on surface of
the APC
INFUSE PATIENT
Active T-cell
Inactive T-cell
T-cells proliferate and attack cancer cells
Sipuleucel-T activates T-cells in the body
The precise mechanism of sipuleucel-T in prostate
cancer has not been established.
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Antigen Presentation to T Cells
T-cell
CD8CD4
CD154
CD11/CD18
CD28
TCR
Peptide
MHC class IMHC class II
CD54
CD80CD86
CD40
Antigen Presenting Cell
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Autologous Cellular Immunotherapy Product Testing

• Challenges
• Heterogeneous starting material
• Limited patient materials
• HLA-restricted APC activity
• Unknown patient HLA haplotypes
• Short product shelf life
• Bioassays are difficult to validate

• Solutions
• Evaluate healthy donor cells as a model for
  patient cells
• Characterize the product and process for
  uniformity and control
• Identify target cells responsible for antigen
  presentation to T cells
• Correlate target cell phenotype and antigen
  presentation activity
• Develop assays that can be validated and related
  to clinical outcome

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Cell Product Characterization Tools

• Healthy HLA-phenotyped donor cells obtained from
  apheresis
• Fluorescently labeled monoclonal antibodies,
  commercially available
• Fluorescently labeled recombinant antigen
  (PA2024-FITC)
• 2 PAPHLA DR1-specific T cell hybridoma lines
• Patient cells evaluated as part of product
  release

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Correlation CD54 and CD14 in Final Product
Healthy Donors and Clinical Trial Patients
2500
Healthy Donor
FP CD14
9902B - FP CD14
D9902B
y 0.9321x - 36.425
y 0.8531x - 20.09
Linear (HD)
2
2
R
0.9681
R
0.8323
Linear (D9902B)
2000
1500
CD14 Cell Count
1000
500
0
0
500
1000
1500
2000
2500
CD54 Cell Count
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In-vitro T Cell Activation Correlates with
Upregulation of Co-stimulatory Molecules on APCs
Post-culture
Pre-culture
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PA2024-FITC is Taken Up by CD54 APCs
HLA-DR
CD54
CD40
PA2024-FITC
CD14
CD19
CD3
PA2024-FITC
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Potency Assay Overview

• Number of CD54 cells (as measured by flow
  cytometry and cell count)
• CD54 fold-upregulation (as measured by flow
  cytometry before/after culture with PA2024
  antigen)
• Flow cytometry method utilizes
• Commercially available fluorescently labeled
  antibodies
• Commercially available fluorescently labeled
  calibration bead standards
• Standardized operating procedures
• Flow cytometry method is
• Reproducible and robust
• Linear over the range of values
• Validatable

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Antigen Presentation AssayHLA-Restricted, for
Product Characterization Only
Mouse T Cell Hybridoma
CD4
TCR
PAP Peptide
HLA DR-1
Human Antigen Presenting Cell
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Antigen Presentation Tracks with PA2024-FITC
Uptake
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Antigen Presentation Activity Tracks with CD54
Cells
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Antigen Presentation Decays with Time and
Temperature Though the assay is somewhat
variable and difficult to validate
RAPA
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CD54 Cell Mean Fluorescence Intensity (MFI) is
Stability-indicating90 prediction limit analysis
Normal Storage
Stressed Conditions
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Box and Whisker plots
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CD54 Cell Numbers are Comparable for All Phase
3 Studies
D9901 D9902A
D9902B P-11
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CD54 Upregulation Ratios are Comparable for All
Phase 3 Studies
D9901 D9902A
D9902B P-11
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Pooled K-M Survival Curves Cumulative CD54 Cell
Dose Above vs. Below the Median for
Sipuleucel-T-treated Patients, Compared with
Placebo-treated Patients
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Pooled K-M Survival Curves for Sipuleucel-T-treate
d Patients Cumulative CD54 Upregulation Above
vs. Below the Median, Compared with Placebo
Patients
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Summary of Sipuleucel-T Potency Testing

• Healthy donor cells mimic clinical data for CD54
  expression and upregulation
• PAP-specific HLA DR1-restricted T cell hybridomas
  demonstrate antigen presentation activity in
  healthy donor and patient cells
• PAP-specific antigen presentation activity
  resides with CD54 cells
• CD54 expression and upregulation appear to be
  surrogates for PAP-specific antigen presentation
  activity
• CD54 expression is stability-indicating
• CD54 expression and upregulation may correlate
  with survival
• CD54 cell count and CD54 upregulation are
  biologically relevant potency measures, as part
  of a matrix of release tests (including
  viability, total cell count, and PAP-specific
  identity)

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