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Potency Testing for an Autologous Cellular Immunotherapy

Potency Testing for an Autologous Cellular Immunotherapy. Nicole Provost, PhD VP Product Development Dendreon Corporation February 9, 2006. Overview. Introduction to the process and product Model system – healthy donor apheresis cells Molecular tools and cellular assays

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Potency Testing for an Autologous Cellular Immunotherapy

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  1. Potency Testing for an Autologous Cellular Immunotherapy Nicole Provost, PhD VP Product Development Dendreon Corporation February 9, 2006

  2. 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

  3. Sipuleucel-T (Provenge®)Manufacturing Process Day 1 Leukapheresis Day 2-3 Sipuleucel-T is manufactured Day 3-4 Patient is infused Apheresis Center Dendreon Doctor’s Office COMPLETE COURSE OF THERAPY: 3 CYCLES

  4. Cellular Immunotherapy with Sipuleucel-T Recombinant Prostatic Acid Phosphatase (PAP) antigen combines with resting antigen presenting cell (APC) APC takes up the antigen Antigen is processed and presented on surface of the APC Fully activated, the APC is now sipuleucel-T 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.

  5. 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

  6. 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

  7. 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 PAP+HLA DR1-specific T cell hybridoma lines • Patient cells evaluated as part of product release

  8. 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

  9. In-vitro T Cell Activation Correlates with Upregulation of Co-stimulatory Molecules on APCs Post-culture Pre-culture

  10. PA2024-FITC is Taken Up by CD54+ APCs HLA-DR CD54 CD40 PA2024-FITC CD14 CD19 CD3 PA2024-FITC

  11. 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

  12. Antigen Presentation Assay:HLA-Restricted, for Product Characterization Only Mouse T Cell Hybridoma CD4 TCR PAP Peptide HLA DR-1 Human Antigen Presenting Cell

  13. Antigen Presentation Tracks with PA2024-FITC Uptake

  14. Antigen Presentation Activity Tracks with CD54+ Cells

  15. Antigen Presentation Decays with Time and Temperature ……Though the assay is somewhat variable and difficult to validate 1200 Normal Storage Stressed Conditions 1000 800 Antigen Presenting Activity 600 400 200 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) % RAPA

  16. CD54+ Cell Mean Fluorescence Intensity (MFI) is Stability-indicating90% prediction limit analysis Normal Storage Stressed Conditions

  17. Box and Whisker plots

  18. CD54+ Cell Numbers are Comparable for All Phase 3 Studies D9901 D9902A D9902B P-11

  19. CD54 Upregulation Ratios are Comparable for All Phase 3 Studies D9901 D9902A D9902B P-11

  20. Pooled K-M Survival Curves: Cumulative CD54 Cell Dose Above vs. Below the Median for Sipuleucel-T-treated Patients, Compared with Placebo-treated Patients

  21. Pooled K-M Survival Curves for Sipuleucel-T-treated Patients: Cumulative CD54 Upregulation Above vs. Below the Median, Compared with Placebo Patients

  22. 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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