# Presentation: 358190

## 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 Center

- Dendreon

- Doctor’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**

## Antigen Presentation to T Cells

- T-cell

- Antigen Presenting Cell

- TCR

- MHC class IMHC class II

- CD8CD4

- CD80CD86

- CD28

- CD54

- CD11/CD18

- Peptide

- CD154

- CD40

## 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 = 0.9321x - 36.425

- R

- 2

-  = 0.9681

- 9902B - FP CD14

- y = 0.8531x - 20.09

- R

- 2

-  = 0.8323

- 0

- 500

- 1000

- 1500

- 2000

- 2500

- 0

- 500

- 1000

- 1500

- 2000

- 2500

**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

## Antigen Presentation Assay:HLA-Restricted, for Product Characterization Only

- 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**

## 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

- 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

- 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)