Transcript Potency Assays For Recombinant Viral Vaccines For Cancer Therapy Kelledy Manson

Potency Assays For Recombinant Viral
Vaccines For Cancer Therapy
Kelledy Manson
Sr. Director Bioanalytical Development
Therion Biologics Corporation
February 9, 2006
Agenda
• Introduction to Therion products
• Challenges for Therion potency assays
• Matrix approach to potency evaluation
• Surrogate assays
• Questions and Answers
Introduction to Therion Products
• PANVAC-VF
– Phase 3 for the treatment of patients with metastatic
pancreatic cancer
• PROSTVAC®-VF
– Phase 2 for the treatment of patients with metastatic
prostate cancer
Poxviruses deliver genes encoding target tumor
antigens and immune-enhancing proteins
Complex Biologic Function
Complex Biologic Function
Poxvirus Vectors
• Vaccinia (V)
– Replicates in multiple cell types
– Replication limited by rapid host immune response
• Fowlpox (F)
– Replicates only in avian cells
– Infects and expresses proteins but does not replicate in
mammalian cells
– Protein expression not inhibited by host immune
response to fowlpox
Administered in a heterologous prime-boost regimen
V F F F ···
Vaccines Express Tumor-Associated
Antigens and Costimulatory Molecules
• Tumor-Associated Antigens (TAA)
– PANVAC-VF
• CEA
• MUC-1
– PROSTVAC®-VF
• PSA
• TRIad of COstimulatory Molecules -TRICOM™
– B7.1
– LFA-3
– ICAM-1
Therion Biological Potency Assays
• Measure biological activity that relates to
product function
• Ensure lot-to-lot consistency
• Stability-indicating
• Minimize variability
• Include suitable reference standards
Challenges for Therion Potency Assays
• Complex products with multiple
components
–Two distinct viral vectors with different
biological characteristics
–One or two tumor-associated antigens
–Human costimulatory molecules
• Series of events required for biological
activity
Statistical Approach to Biological Assay
Development
• Statistical design of experiments
• Identification of the sources of assay variability
• Informs the number of replicates or doses for
accuracy and precision
• May lead to assay modifications and refinements
• Accommodates use of multiple lots of reference
standard
Evolution of Therion Potency Assays
DEVELOPMENT STAGE
ASSAY TYPE
Plaque titration
Phase 1-2 Trials
Biological activity
Phase 3 Trials
MATRIX APPROACH
Critical steps required
for biological function
Approved Product
Quantitative Surrogate
Complex Biologic Function
BIOLOGICAL ACTIVITY
PROTEIN EXPRESSON
GENETIC CODING
INFECTION
Summary of Matrix Assays to Measure
Critical Steps
Function
Parameter Measured
QC Release Test
Infection
Number of infectious virus particles in
Drug Product
Plaque titration assay
Genetic Coding
Genetic structure encoding TAAs and
TRICOM
Southern blot analysis
Expression of proteins
Western blot analysis
Percentage of virus particles that
express proteins
In situ immunoassay
TAA expression and immune
responses in mice
In vivo potency assay
Ability of TRICOM molecules to
activate human T cells
In vitro T cell activation
assay
Protein Expression
Biological Activity
How is TRICOM Designed to Work?
SIGNAL 1
TRICOM
SIGNAL 2
Activation of
Antigen-Specific T Cells
The In Vitro TRICOM Assay
• Activation of human T cells in vitro
–Signal 1: Non-specific
–Signal 2: Poxvirus-infected cell line expressing
TRICOM
• T cell activation quantitated by
measurement of cytokine secretion
Design of Experiments for In Vitro TRICOM
Assay
• Sources of variability in a single experimental
design:
– Day-to-day
– Operator-to-operator
– Naïve T cell donor source
– Assay plate-to-plate
– Well-to-well
– ELISA plate to ELISA plate
• Dose-response to different multiplicities of infection
• Designed to increase sensitivity to allow
assessment of lot-to-lot consistency and stability
Surrogate Assays
Evaluating surrogate assays for biological function:
• Analytical
• Quantitative
• More reproducible
• Correlation with biological activity (e.g., use of
quantitative flow cytometry to replace in vivo
potency assay)
Summary
• Complex products require more than one assay to
assess activity
• Matrix of analytical assays can be used to evaluate
critical steps
• Statistical approach for biological activity assays is
critical for development and validation
• Quantitative surrogate assays can replace or
supplement variable biological assays for
consistency and stability assessment
Potency Assays For Recombinant Viral
Vaccines For Cancer Therapy
Kelledy Manson
Sr. Director Bioanalytical Development
Therion Biologics Corporation
February 9, 2006