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

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Presentation transcript:

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 Correlating antigen presentation activity with cell phenotype Justifying potency assays Tracking potency over time Comparing potency data with clinical outcomes Q & A

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

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 The precise mechanism of sipuleucel-T in prostate cancer has not been established. Antigen is processed and presented on surface of the APC INFUSE PATIENT T-cells proliferate and attack cancer cells Sipuleucel-T activates T-cells in the body Active T-cell Inactive T-cell

T-cell Antigen Presenting Cell TCR MHC class I MHC class II CD8 CD4 CD80 CD86 CD28 CD54 CD11/CD18 Peptide CD154 CD40 Antigen Presentation to T Cells

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

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

Correlation: CD54 and CD14 in Final Product Healthy Donors and Clinical Trial Patients FP CD14 y = x R 2 = B - FP CD14 y = x R 2 = CD54 Cell Count CD14 Cell Count Healthy Donor D9902B Linear (HD) Linear (D9902B)

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

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

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

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

Antigen Presentation Tracks with PA2024-FITC Uptake

Antigen Presentation Activity Tracks with CD54+ Cells

Antigen Presentation Decays with Time and Temperature … …Though the assay is somewhat variable and difficult to validate % RAPA Time (hrs) Antigen Presenting Activity Stressed Conditions Normal Storage

CD54+ Cell Mean Fluorescence Intensity (MFI) is Stability-indicating 90% prediction limit analysis Normal Storage Stressed Conditions

Box and Whisker plots

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

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

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

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

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)