Standardization of MHC Tetramer and ELISPOT Assays to Quantitate -Specific Expansion after CTLA4 Blockade

A. Ribas1, B. Comin-Anduix1, J. A. Glaspy1, R. Renteria2, D. Reardon3, E. Seja1, B. Englahner3, M. Ontiveros1, J. S. Economou1, B. Littman3, J. Gomez-Navarro3, A. Gualberto3

1University of California Los Angeles, Los Angeles, CA 2Beckman Coulter, Inc., San Diego, CA 3Pfizer Global Research and Development, New London, CT Background and Study Goal

• Background: – Implementation of cellular immunological assays to monitor immunotherapy require quality control monitoring and assay validation studies (Keilholz et al. JIT 2002).

•Goal: – Define a “positive immunological response” to immunotherapy taking into account the assay imprecision. Performance Specifications: LLD and Assay Imprecision

LLD = Mean of a Negative Epitope + 2 Standard Deviations

Standard Deviation Coefficient of Variation (CV) = x 100 Mean

Total Assay Variation = preCV2 + aCV2 + pCV2 preCV = Pre-analytical variation: - Sample collection. Minimized by standardized - Sample processing. sample collection and processing, - Storage conditions. and adherence to SOP of - Assay conditions. optimized assays. aCV = Analytical variation. pCV = Physiological variation. How can we define a “positive immunological response” taking into account the assay imprecision?

1. Minimize pre-analytical variability with assay optimization and SOPs.

2. Reference Change Value (RCV) = 2 x Z x aCV2 + pCV2

Z = Two-tailed Z score with 0.05% significance. aCV = Analytical variation. pCV = Physiological variation. Analytical CV

Run MHC Tetramer or ELISPOT

Run MHC Tetramer or ELISPOT

Run MHC Tetramer or ELISPOT

• Goal: Determine the inherent assay variability running replicate samples. • Method: Analyze replicate samples with values above the LLD from the same blood draw re-run at different time points. Physiological CV

Day 1 Run MHC Tetramer and ELISPOT

Day 2-7 Run MHC Tetramer and ELISPOT

Day 14-21 Run MHC Tetramer and ELISPOT

Day 30-50 Run MHC Tetramer and ELISPOT

• Goal: Determine the changes over time in baseline levels of circulating antigen-specific T cells. • Method: Analyze samples from different blood draws taken within a period of 2 months without any treatment. • 121 blood draws to 31 subjects; 90% (28 subjects ) had the 4 scheduled blood draws. Subject Eligibility

• Healthy Donors (10): – HLA-A*0201. • Patients with Melanoma (21): – HLA-A*0201. – No prior therapy within 30 days. – No therapy during blood procurements (1-2 mo.). – Stage II-IV melanoma. – MART-1 positive by IHC or RT-PCR. – No concurrent immunosuppressive therapy. Patients with Melanoma No Evidence of Disease (NED) Alive with Disease (AWD) Stage Trial Prior Therapy Stage Trial Prior Therapy No. No. II 107 MART1/DC III 102 None III 105 None 103 Chemotherapy, MART1/DC, 111 IFN- α, MART1/DC, CP-675,206 CP-675,206 IV 101 None IV 100 MART1/DC 104 None 108 None 106 MART1/DC, CP-675,206 109 MART1/DC, CP-675,206 112 MART1/DC, CP-675,206 110 BCG, MART1/DC, CP-675,206 114 CP-675,206 113 MART1/DC 116 CP-675,206 115 IL-2, MART1/DC, CP-675,206 117 Chemotherapy, MART1/DC 118 Chemotherapy, IL-2, IFN-α, CP-675,206 119 Chemotherapy, MART1/DC, In all cases, prior MART1/DC and CP-675,206 had CP-675,206 been completed over 3 months from enrollment. 120 None Tetramer Assay Optimization

• Tetramer source: BC > NIAID. • Flow cytometer monitoring: Daily FlowSet fluorospheres. • Gating: Initial gating out non-CD8 cells (BC approach) > Initial gating on lymphocytes by size (consensus workshop approach, Keilholz et al. JIT 02). • Number of CD8+ events: 30-200,000. MHC Tetramer Low Limit of Detection (LLD)

Epitope No. Mean SD LLD Samples Negative 121 0.02 0.01 0.03 FLYSYFALV1 2 AFP325-332 119 0.05 0.03 0.11

1. Ruppert, J., et al. Cell 1993;74:929-937. 2. Butterfield, L.H., et al. Journal of 2001; 166: 5300-5308. Analytical CV MHC Tetramer Assay

Epitope No. Mean SD aCV Samples EBV 26 3.19 0.26 8%

BMLF1259-267

CMVpp65495-503 10 0.37 0.04 10%

MART-126-35 66 5.29 0.44 8%

5-8 replicate aliquots of the same cryopreserved samples. Results over the LLD for the assay. CMVpp65495-503 Tetramer Staining

8.00

7.00

6.00

5.00

4.00 of Tetramer positive cells of Tetramer e 3.00

2.00 Percentag

1.00

0.00

6 3 3 8 7 2 1 8 9 5 6 8 0 3 5 0 2 0 4 9 9 1 7 4 0 0 # # 1 #6 #4 #2 #5 #1 # 1 1 #7 10 11 12 11 #1 11 11 #1 # #10 #11 #10 #10 #10 #10 #1 # # # #11 #11 #10 #10 #1 #1 # # #10

CMV Serology Negative Positive Physiological CV

CMVpp65495-503 Tetramer Assay

All samples: Clinical Group No. Samples Mean SD P Value pCV

Seronegative 73 0.08 0.03 41%

Seropositive 47 0.87 0.07 0.012 8%

Samples over the LLD for the assay:

Clinical Group No. Samples Mean SD pCV

Seronegative 69 0.08 0.03 41%

Seropositive 47 0.87 0.07 8% EBV BMLF1259-267Tetramer Staining

5.50

5.00

4.50

4.00

3.50

3.00

of Tetramer positive cells of Tetramer 2.50 e 2.00

1.50 Percentag 1.00

0.50

0.00 #3 #2 #100 #9 #120 #116 #111 #106 #5 #112 #113 #107 #109 #110 #6 #114 #108 #115 #102 #1 #119 #117 #105 #118

EBV Serology Negative Indeterminate Positive Physiological CV

EBV BMLF1259-267 Tetramer Assay All samples: Clinical Group No. Samples Mean SD P Value pCV

Seronegative 43 0.52 0.18 36%

Indeterminate 27 1.53 0.52 0.004 34%

Seropositive 24 1.08 0.14 0.005 13%

Samples over the LLD for the assay: Clinical Group No. Samples Mean SD pCV

Seronegative 43 0.52 0.18 36%

Indeterminate or 51 1.32 0.35 26% Seropositive MART-126-35 Tetramer Staining

6.00

5.00

4.00

3.00 of Tetramer positive cells of Tetramer e

2.00 Percentag

1.00

0.00

5 8 4 0 7 5 3 9 4 0 # #2 #1 # #3 # 1 #6 # #9 17 00 0 07 11 1 15 10 0 02 16 18 03 01 19 14 0 06 12 2 08 # 1 1 1 1 1 1 1 1 1 1 1 #1 #1 # #1 # #1 #1 # #1 # # #1 # # #1 # # #1 # # #1 Healthy Subjects NED AWD Patients with Melanoma Subject #109: Circulating MART-126-35-Specific CD8+ T Cells Over 6 Months

11/30/2002 12/23/2002 05/05/2003 + 7.73% 7.38% 6.32% Tetramer 26-35 MART-1

CD8+ Physiological CV

MART-126-35 Tetramer Assay All samples: Clinical Group No. Samples Mean SD P Value pCV Healthy Donors 39 0.21 0.05 24

NED 33 1.00 0.33 0.01 33

Active Melanoma 47 0.13 0.03 0.33 20

Samples over the LLD for the assay: Clinical Group No. Samples Mean SD pCV Healthy Donors 35 0.23 0.05 24

NED 33 1.01 0.32 32

Active Melanoma 31 0.19 0.04 19 IFN-γ ELISPOT Assay Optimization

• APC: K562/A*0201 > T2 > JY. • APC/PBMC Ratio: 1:10 > 1:2. • Peptide-pulsing Titration: 10 µg > 50 µg. • No. PBMC per Well: 1 x 105 > 2 x 105. • PBMC Restimulation: In plate > In culture flask.

• Spot-forming Cells: CD8 > NK > CD4. • A*0201 phenotyping of K562/A*0201 cells: > 96%. • Mycoplasma contamination of K562/A*0201 cells: Negative IFN-γ ELISPOT Low Limit of Detection (LLD)

Epitope No. Mean SD LLD Samples Negative 411 3 1 6 FLYSYFALV1

2 AFP325-332 359 6 1 8

1. Ruppert, J., et al. Cell 1993;74:929-937. 2. Butterfield, L.H., et al. Journal of Immunology 2001; 166: 5300-5308. Analytical CV IFN-γ ELISPOT Assay No. Epitope Mean SD aCV Samples

EBV BMLF1259-267 24 77 12 16%

CMVpp65495-503 ----

MART-127-35 61 27 18 27%

MART-126-35 50 176 39 22% 7-9 replicate aliquots of the same cryopreserved samples. Only samples over the LLD for the assay. CMVpp65495-503 IFN-γ ELISPOT

250

200 Cells 5

150

100 No. Spots/10

50

0

3 7 5 2 6 5 0 9 #3 #8 06 0 08 #4 #6 01 13 1 18 09 10 20 #2 02 #1 #5 04 #9 11 #7 14 17 00 10 10 11 11 #1 11 # #1 #1 #1 #1 # # #1 #1 #1 #1 #1 #1 # #1 #1 # #1 #1 #1 #1

CMV Serology Negative Positive Physiological CV

CMVpp65495-503 ELISPOT Assay

All samples: Clinical Group No. Samples Mean SD P Value pCV

Seronegative 219 4 2 37%

Seropositive 132 32 3 < 0.0001 11%

Samples over the LLD for the assay:

Clinical Group No. Samples Mean SD pCV

Seronegative 46 16 4 28%

Seropositive 77 49 5 10% EBV BMLF1259-267 IFN-γ ELISPOT

250

200 Cells

5 150

100 No. Spots/10 50

0

6 8 2 0 #3 13 #5 00 #2 16 11 #6 20 12 07 09 14 10 15 #1 05 19 17 18 10 10 #1 #1 #1 #1 #1 #1 #1 #1 #1 #1 # #1 #1 # #1 #1 #1 #1

EBV Serology Negative Indeterminate Positive Physiological CV

EBV BMLF1259-267 ELISPOT Assay All samples: Clinical Group No. Samples Mean SD P Value pCV

Seronegative 147 10 3 33%

Indeterminate 73 34 5 0.015 16%

Seropositive 75 32 5 < 0.0001 17%

Samples over the LLD for the assay: Clinical Group No. Samples Mean SD pCV

Seronegative 49 23 7 29%

Indeterminate or 103 47 8 18% Seropositive MART-127-35 IFN-γ ELISPOT

200

150 Cells 5

100

No. Spots/10 50

0

1 2 8 9 4 0 5 7 3 5 0 7 9 1 5 0 3 4 1 2 6 8 2 6 9 4 8 0 # # #3 # # # #6 # #7 0 0 1 0 0 0 0 1 #1 11 11 10 10 11 11 11 #1 # #1 #1 #11 #10 # #11 #1 # #1 #1 #10 #1 # #11 # # #1 # #12

NED AWD Healthy Subjects Patients with Melanoma Physiological CV MART- 127-35 ELISPOT Assay All samples: Clinical Group No. Samples Mean SD P Value pCV Healthy Donors 118 1 1 62%

NED 108 17 2 0.0045 14%

Active Melanoma 138 6 2 0.0015 32%

MART-126-35 Active Melanoma 53 13 4 34%

Samples over the LLD for the assay: Clinical Group No. Samples Mean SD pCV Healthy Donors - - - -

NED 46 37 5 12%

Active Melanoma 22 28 7 24%

MART-126-35 Active Melanoma 32 18 6 31% Reference Change Value (RCV)

• After minimization of pre-analytical variability, the assay imprecision has: – Random error following a Gaussian distribution. – Components of assay variability: aCV and pCV.

• RCV: Allows to define which change from baseline to post immune intervention would be beyond the assay variability. Reference Change Value (RCV) for Tetramer Assay

Epitope No. aCV No. pCV Z RCV Samples Samples score aCV pCV

EBV BMLF1259-267 26 8 24 13 2.09 36%

CMVpp65495-503 10 10 27 9 2.10 30%

MART-126-35 NED 66 8 34 33 2.01 75%

MART-126-35 66 84820 2.01 47% Active Melanoma

RCV = 2 x Z x aCV2 + pCV2 Reference Change Value (RCV) for ELISPOT Assay

Epitope No. aCV No. pCV Z RCV Samples Samples score aCV pCV

EBV BMLF1259-267 24 16 72 17 2.01 55%

CMVpp65495-503 - -1114 1- -

MART-127-35 NED 61 27 33 9 2.01 68%

MART-127-35 61 27 28 24 2.01 83% Active Melanoma

MART-126-35 50 22 34 31 2.02 90% Active Melanoma

RCV = 2 x Z x aCV2 + pCV2 Exploratory Study of Antigen-Specific Immune Responses in the First-in-Human CP-675,206 Study (anti-CTLA4 mAb)

• 3 HLA-A2.1+ patients agreed to donate extra blood for immune monitoring at redosing.

• CP-675,206 redosing for the following reasons: – 2 previously enrolled in a lower dose cohort. – 1 for clinical benefit. Phase I Trial No. 1001 113, Redosed at 3 mg/kg: Tetramer Assay Pre-αCTLA-4 d+7 d+33 d+41

4 4 4 4 10 10 10 10 R4 R4 R4 R4 0.02 0.01 0.00 0.01 3 3 3 3 10 10 10 10 H H H Negative H 2 2 2 2 L2- L2- L2- L2- 10 10 10 10 F F F F

1 1 1 1 10 10 10 10

0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL1-H FL1-H FL1-H FL1-H

4 4 4 4 10 10 10 10 R4 R4 R4 R4 4.01 3.78 3.56 2.69 3 3 3 3 10 10 10 10 H H H H - -

EBV 2 2 2 2 2 2 L2- 10 L2- 10 10 10 FL FL F F

1 1 1 1 10 10 10 10

0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL1-H FL1-H FL1-H FL1-H

4 4 4 10 10 4 10 R4 R4 10 R4 0.07 0.05 0.07 R4 0.09 3 10 3 3 3 10 10 10 H H H H 2 -

2 2 2 2 L2- 10

10 L2- L2- 10 10

MART-1 F FL F F

1 10 1 1 1 10 10 10

0 10 0 0 0 0 1 2 3 4 10 10 10 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL1-H + FL1-H FL1-H CD8 FL1-H Phase I Trial No. 1001 102, Redosed at 6 mg/kg: Tetramer Assay Pre-αCTLA-4 d+2 d+27 d+41 d+80 Negative 0.02 0.01 0.01 0.02 0.01

EBV 0.84 1.14 1.46 0.95 0.86

↑36% ↑74% (RCV = 63%) MART-126-35 0.02 0.03 0.04 0.05 0.04

↑250% (RCV = 47%) CD8+ Phase I Trial No. 1001 107, Redosed at 6 mg/kg: Tetramer Assay Pre-αCTLA-4 d+26 d+51 d+65

0.01 R4 0.03 R4 0.02 R4 0.03 R4 Negative

0.42 R4 0.36 R4 0.45 R4 0.40 R4

EBV

R4 0.20 R4 0.43 0.22 R4 0.18 R4 MART-1

(RCV = 47%) CD8+ ↑115% Conclusions • A 50% increase in circulating MART-1-specific T cells using MHC tetramers is beyond the assay variability and should be considered a positive response to immunotherapy. • An 80% increase in IFN-γ-producing MART-1- specific T cells using ELISPOT is beyond the assay variability and should be considered a positive response to immunotherapy. • In a preliminary analysis, CTLA4 blockade with the monoclonal antibody CP-675,206 can increase the frequency of circulating antigen-specific T cells. Acknowledgements

•UCLA: • Pfizer GR&D: – Begonya Comin-Anduix, PhD – Antonio Gualberto, MD, PhD – Elisabeth Seja – Deborah Reardon – Maribel Ontiveros – Brigitte Englahner – Rosalinda Rivera – Bruce Littman, MD – Jackie Hernandez – Jesus Gomez-Navarro, MD – Denise Oseguera • Beckman Coulter – John A. Glaspy, MD, MPH Immunomics: – James S. Economou, MD, PhD – Roberto Renteria