Potency Assays for an Autologous Active Immunotherapy (Sipuleucel-T) Pocheng Liu, Ph.D. Senior Scientist of Product Development Dendreon Corporation

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1 Potency Assays for an Autologous Active Immunotherapy (Sipuleucel-T) Pocheng Liu, Ph.D. Senior Scientist of Product Development Dendreon Corporation

2 Sipuleucel-T 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

3 Cellular Immunotherapy with sipuleucel-t PA224 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.

4 Antigen Presentation Assay (Hybridoma Assay) Mouse T Cell Hybridoma (Papillon, Paperino) TCR CD4 IL-2 PAP Peptide HLA DR-1 Human Antigen Presenting Cell (Sipuleucel-T)

5 Challenges for Autologous Cellular Immunotherapy Product Potency Testing Challenges Heterogeneous and limited final product Introduce assay variability No reference material HLA-restricted APC activity Varied patient HLA haplotypes Short product shelf life Long bioassay incubation time Solutions Surrogate potency assay Identify target cells responsible for antigen presentation to T cells Correlate target cell phenotype and antigen presentation activity Develop assays that can be used for product potency test Reproducible, robust Universal to all patient Short incubation time Tracking assay over time Confirm potency data with clinical outcome.

6 Antigen Presentation Tracks with PA224-FITC Uptake Tritiated thymidine uptake (cpm) 3, 25, 2, 15, 1, 5, FITC +ve FITC -ve Papillon 7, 6, 5, 4, 3, 2, 1, Paperino No. of input cells

7 PA224-FITC is Taken Up by CD54+ APCs CD54 HLA-DR CD4 PA224-FITC CD3 CD19 CD14 PA224-FITC

8 Antigen Presentation Activity Tracks with CD54+ Cells Tritiated thymidine uptake (cpm) 25, 2, 15, 1, 5, CD54 +ve CD54 -ve Papillon 5, 4, 3, 2, 1, Paperino No. of input cells

9 Upregulation of Co-stimulatory Molecules on APCs Pre-culture Post-culture CD54 5, 4, 3, 2, CD56 1, CD4 HLA-DR

10 Surrogate Potency Assays Quantity of APC Total number of CD54+ cells Measured by flow cytometry and total nucleated cell count Quality of APC CD54 up-regulation Measured CD54 expression by flow cytometry before/after culture with PA224 antigen

11 CD54 Up-regulation Ratio Before Culture After Culture CD54(+) A BDS65.7 Large CD54 + Cells IgG2b + Cells A FP-Control.7 Large CD54 + Cells IgG2b + Cells FSC-Height A BDS FSC-Height A FP-Control.7 File: A BDS65.7 Sample ID: Patient ID: Acquisition Date: 5-Jun Gate: G4 CD54 PE Events Geo Mean File: A FP-Control.7 Sample ID: Patient ID: Acquisition Date: 7-Jun-6 Gate: 1 G CD54 PE Events Geo Mean

12 Potency Assay Overview Flow based assays to quantify total CD54+ cells and changes in CD54 expression before and after incubation Not limited by HLA haplotypes Short assay incubation time Reproducible and robust Commercially available fluorescence standard

13 Antigen Presentation Decays under a Stressed Condition Normal Storage Stressed Condition 12% 1% RAPA Decay A2-345 A A % 1% RAPA Decay A A A % of Time 8% 6% 4% % of Time 8% 6% 4% 2% 2% % Time % Time

14 Total Number of CD54+ Cells Decays under a Stressed Condition Normal Storage Stressed Condition 12% 1% Total CD54+ cells Decay A2-345 A A % 1% Total CD54+ Cell Decay A A A % of Time 8% 6% 4% % of Time 8% 6% 4% 2% 2% % % Time Time

15 CD54 Expression Decays under a Stressed Condition Normal Storage Stressed Condition 12% CD54 Expression Decay A2-345 A A % CD54 Expression Decay A A A % 1% % of Time 8% 6% 4% % of Time 8% 6% 4% 2% 2% % % Time Time

16 K-M Survival Curves: Cumulative CD54 Cell Dose Above vs. Below the Median Comparison sipuleucel-t ( 2.3 x 1 9 cells; n=73) Percent survival sipuleucel-t (< 2.3 x 1 9 cells; n=73) p=.237 Placebo (n=76) Survival Time (months)

17 K-M Survival Curves: Cumulative CD54 Up-regulation Above vs. Below the Median Comparison Percent survival sipuleucel-t ( 22.3; n=73) sipuleucel-t(< 22.3; n=73) p=.6 Placebo (n=76) Survival Time (months)

18 Conclusions PAP-specific HLA DRβ1-restricted T cell hybridoma demonstrates antigen presentation activity in sipuleucel-t cells. PAP-specific antigen presentation activity resides with CD54+ cells. CD54 up-regulation and total CD54+cells are biologically relevant potency measures. Antigen presentation and CD54 up-regulation assays can be stability indication assays. CD54 up-regulation may correlate with survival.

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20 Overview Sipuleucel-T: Investigational active immunotherapy for prostate cancer Potency assay challenges for autologous cellular products Model system: Antigen presentation assay Surrogate markers Correlating antigen presentation and other biological activity Tracking potency assay Confirmation: Comparing potency assay with clinical outcome

21 Correlation: CD54 and CD14 in Final Product Healthy Donors and Clinical Trial Patients 25 FP CD14 y =.9321x R 2 = B - FP CD14 y =.8531x R 2 =.8323 Healthy Donor D992B Linear (HD) Linear (D992B) 2 CD14 Cell Count CD54 Cell Count

22 Antigen Presentation Dose Response Curve OD Log1(FP/mL) Reference Test

23 Relative Antigen Presentation Activity (RAPA) Log1(OD) Log1(FP/mL) Reference Test Fitted Ref Fitted Test

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

25 Assay Characterization %CD54 cells # of CD54 molecules Intra-assay precision <7% <4% Inter-assay precision <6.5% <13.5% Lab-to-Lab variability <2% <9%

26 Fluorescence Standard < 7 Med - High High Med - Low Low File: /4/99.1 X Parameter: FL2-H CD54 PE 1:1 (Log) Marker Events Geo Mean CV All Low Med - Low Med - High High > 7 Calibration curve Slope: Intercept: # PE molecules per bead # PE molecules Geometric means per bead of bead peaks