Potency Testing for an Autologous Cellular Immunotherapy

Nicole Provost, PhDVP Product DevelopmentDendreon CorporationFebruary 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 1Leukapheresis

Day 2-3Sipuleucel-T is manufactured

Day 3-4Patient 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 APCINFUSE 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

TCRTCR

MHC class IMHC class IMHC class IIMHC class II

CD8CD8CD4CD4

CD80CD80CD86CD86

CD28CD28

CD54CD54

CD11/CD18CD11/CD18

Peptide

CD154CD154

CD40CD40

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 CD14y = 0.9321x - 36.425

R2 = 0.9681

9902B - FP CD14y = 0.8531x - 20.09

R2 = 0.8323

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

CD54 Cell Count

CD

14 C

ell

Co

un

t

Healthy Donor

D9902B

Linear (HD)

Linear (D9902B)

(a) Allogeneic T cell stimulation

0

4

8

12

16

20

24

28

0 3.2 6.4 12.5 25 50 100 200 400

Number of APC8015 cells (x103/well)

3H

-Th

ym

idin

e I

nc

orp

ora

tio

n (

x1

04

cp

m)

Pre-culture

Post-culture

(b) Autologous T cell stimulation

0

2

4

6

8

10

12

0 3.2 6.4 12.5 25 50 100 200 400

Number of APC8015 cells (x103/well)

3H

-Th

ym

idin

e I

nc

orp

ora

tio

m (

x1

03 c

pm

) Pre-culture

Post-culture

50,000

0.2 0.4 0.8 1.6 3.1 6.3 12.5 25

Number of APC8015 cells per well ( x10 )3

Pre Culture

Post Culture

40,000

30,000

20,000

10,000

0

Increased naïve T cell stimulationactivity after two day in vitro incubation.Peripheral blood mononuclear cells weresingle cell sorted before and after culturefor 40 hours. Cells were processedaccording to standard procedures for theproduction of DC and stained with a PE -labeled CD54 reactive monoclonalantibody. After sorting cells had greaterthan 98% purity. CD54 positive cellpopulation had a high forward and sidescatter profile. Purified CD45RA+ CD4+

T lymphocytes (5x104 cells per well)were mixed with various numbers ofCD54 sorted APC. Five days latertritiated thymidine was added andincorporation determined to generate Tcell proliferation dose response curve.

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

CD5

4 CD40

HLA-D

R

CD

3 CD1

9

PA2024-FITC

CD1

4

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

TCRTCR

HLA DR-1HLA DR-1

CD4CD4

PAP Peptide

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

Antigen Presentation Tracks with PA2024-FITC Uptake

Paperino

100 1000 10000 100000 10000000

25000

50000

75000

FITC+FITC-

APC

cpm

Papillon

100 1000 10000 100000 10000000

10000

20000

30000

APCcp

m

Antigen Presentation Activity Tracks with CD54+ Cells

Papillon

100 1000 10000 100000 10000000

10000

20000

30000

APCcp

m

Paperino

100 1000 10000 100000 10000000

10000

20000

30000

40000

50000

CD54+CD54-

APC

cpm

Antigen Presentation Decays with Time and Temperature ……Though the assay is somewhat variable and difficult to validate

0 5 10 15 20 25 30 35 40 45 500

200

400

600

800

1000

1200

Time (hrs)

An

tig

en P

rese

nti

ng

Act

ivit

y

Stressed Conditions Normal Storage

CD54+ Cell Mean Fluorescence Intensity (MFI) CD54+ Cell Mean Fluorescence Intensity (MFI) is Stability-indicatingis Stability-indicating

90% prediction limit analysis90% prediction limit analysis

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

100110120130

% M

FI o

f Fi

rst

Ass

ay T

imep

oint

Time (hrs)

% M

FI o

f Fi

rst

Ass

ay T

imep

oint

Time (hrs)0 5 10 15 20 25 30 35 40 45 50

0102030405060708090

100110120130

Normal StorageNormal Storage Stressed ConditionsStressed 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)