Clinical Translation of a Mammaglobin Clinical Translation of a Mammaglobin­ ­A A DNA Vaccine for Breast Cancer Therapy DNA Vaccine for Breast Cancer Therapy

William E. Gillanders, M.D. Recombinant DNA Advisory Committee

December 14, 2005

Overview Overview

• This is a phase I dose­ranging vaccine safety trial of a mammaglobin­A DNA vaccine

• The broad objective of the study is to identify a safe and immunologically active dose of the mammaglobin­A DNA vaccine that can be used in future studies

Mammaglobin Mammaglobin­ ­A Tissue Expression A Tissue Expression

Ovary

Uterus

Placenta

Lung

B5­589

Breast

Breast cancer

Watson MA et al, Cancer Research 1996; 56:860

Mammaglobin Mammaglobin­ ­A Tissue Expression A Tissue Expression

Ovary

Placenta

Uterus

Testis

Prostate

PBL

Lymph node

Spleen

Thym

us

Lung

Colon

Bladder

Kidney

Liver

Brain

No template

Breast

Breast C

ancer

GAPDH

hMAM

Watson MA et al, Cancer Research 1996; 56:860

Mammaglobin Mammaglobin­ ­A Tissue Expression A Tissue Expression

Mammaglobin Mammaglobin­ ­A Tissue Expression A Tissue Expression

Watson MA et al, Cancer Research 1999; 59:3028

Mammaglobin Mammaglobin­ ­A Tissue Expression A Tissue Expression

Ductal carcinoma in situ

Poorly differentiated ductal carcinoma

Well differentiated ductal carcinoma

Watson MA et al, Cancer Research 1999; 59:3028

Summary Summary

• Mammaglobin­A is expressed almost exclusively in normal breast epithelium and breast cancer

• Mammaglobin­A is overexpressed in up to 80% of primary and metastatic breast cancers

• Mammaglobin­A overexpression appears to be consistent in all stages of breast cancer

Presentation of Mammaglobin Presentation of Mammaglobin­ ­A A

Jaramillo A et al, International Journal of Cancer 2002; 102:499

0 5 10 15 20 25 30 35 40

No protein

A lbumin

Mammaglobin­A + KuIA2

Mammaglobin­A + W6/32

Mammaglobin­A

Specific Lysis (%)

0 5 10 15 20 25 30

MDA­MB­231

MCF­7

MDA­MB­415

MDA­MB­361

AU­565

HBL­100

Specific Lysis (%)

Mammaglobin Mammaglobin­ ­A A­ ­reactive T cells reactive T cells

0

0.00001

0.00002

0.00003

0.00004

0.00005

CD8+ T cells CD4+ T cells

Frequency (reciprocal) Patients

Controls

Jaramillo A et al, International Journal of Cancer 2002; 102:499

β 2 m Heavy chain

Peptide

MHC Class I trimeric structure MHC Class I trimeric structure

Predicting mammaglobin Predicting mammaglobin­ ­A epitopes A epitopes

Mammaglobin HLA Mammaglobin HLA­ ­A3 peptides A3 peptides

0.30 AIDELKECF 58­66 Mam­A3.8 0.45 LMLAALSQH 07­15 Mam­A3.7 0.60 FLNQTDETL 66­74 Mam­A3.6 1.35 LMVLMLAAL 04­12 Mam­A3.5 1.50 TTNAIDELK 55­63 Mam­A3.4 4.05 KLLMVLMLA 02­10 Mam­A3.3 6.75 KTINPQVSK 31­39 Mam­A3.2 27.00 PLLENVISK 23­31 Mam­A3.1

HLA­A3­binding score

Peptide Sequence

Amino Acid Position Peptide

HLA­A3­Binding Peptides Derived from Mammaglobin­A

Jaramillo A et al, International Journal of Cancer 2002; 102:499

Membrane Stabilization Assay Membrane Stabilization Assay

0 10 20 30 40 50 60 70 80

Influenza

Mam­A3.8 Mam­A3.7

Mam­A3.6 Mam­A3.5 Mam­A3.4

Mam­A3.3 Mam­A3.2

Mam­A3.1

Mean Fluorescence Shift

Jaramillo A et al, International Journal of Cancer 2002; 102:499

Mammaglobin Mammaglobin­ ­A A­ ­reactive T cells reactive T cells

76 0 0 0 0 0 0 0 3 4 109 0 0 7 0 3 7 0 0 3 96 0 0 0 0 0 0 0 0 2 83 3 0 0 0 0 3 0 0 1

Controls 89 0 0 0 0 20 0 0 23 5 103 0 0 3 0 0 57 0 20 4 123 80 7 0 0 183 829 0 0 3 93 23 0 0 0 33 40 0 0 2 127 0 0 0 0 156 0 0 33 1

Influenza A3.8 A3.7 A3.6 A3.5 A3.4 A3.3 A3.2 A3.1 Patients HLA­A3 peptides

Frequency of CD8 T cells reactive to mammaglobin­A­derived peptides in the peripheral blood of HLA­A3 breast cancer patients

Summary Summary

• CD8 and CD4 T cells specific for mammaglobin­A can be generated in vitro from the peripheral blood of breast cancer patients confirming that the immune system can recognize this antigen

• Analyses of peripheral blood from breast cancer patients confirm that breast cancer patients have higher frequencies of mammaglobin­A­reactive T cells

Advantages of DNA Vaccination Advantages of DNA Vaccination

• Safety • Generic manufacture with high purity and stability relative to protein vaccines

• Cost advantage • Vaccination with full­length cDNA avoids the requirement of MHC restriction

•Expression of HLA­A2 on all tissues

•Tissue­specific expression of human CD8 on CD8 + T cells

•Facilitates recognition of human MHC by mouse CD8 + T cells

Humanized Mouse Model Humanized Mouse Model

X C57BL/6 mice transgenic for Human CD8

Human HLA­ A2 + /CD8+

C57BL/6 mice

C57BL/6 mice transgenic for Human HLA­A2

Humanized Mouse Model Humanized Mouse Model

Day 0: DNA vaccine

Day 14: DNA vaccine

Day 7: DNA vaccine

Day 21: DNA vaccine

Day 28: Immune Analysis

Humanized Mouse Model Humanized Mouse Model

Narayanan et al, Journal of the National Cancer Institute 2004; 96:1388

0

10

20

30

40

50

Vaccinated Control A

Breast Cancer Breast Cancer Xenograft Xenograft Model Model

Day 0: Tumor Challenge HBL­100 or MDA­231 cells resuspended in basement membrane

Day 14: Adoptive transfer of 4 x 10 7 spleen cells from vaccinated mice

Tumor size measured by calipers weekly

Breast Cancer Breast Cancer Xenograft Xenograft Model Model

Narayanan et al, Journal of the National Cancer Institute 2004; 96:1388

Phase I Clinical Trial Phase I Clinical Trial

• This is a phase I dose­ranging vaccine safety trial of a mammaglobin­A DNA vaccine

• The broad objective of the study is to identify a safe and immunologically active dose of the mammaglobin­A DNA vaccine that can be used in future studies

Objectives Objectives

• Evaluate the safety of the mammaglobin­A DNA vaccine

• Assess the in vivo immune response induced by the mammaglobin­A DNA vaccine by evaluation of the CD8, CD4 and Treg immune responses

Objectives Objectives

• Assess the impact of mammaglobin­A DNA vaccination on breast cancer tumor markers, including circulating breast cancer cells

• Evaluate enrolled patients for time to disease progression following vaccination with the mammaglobin­A DNA vaccine

Patient Selection Patient Selection

• Patients with stage IV breast cancer are eligible for enrollment

• Eligible patients will have metastatic breast cancer that has been stable for at least 28 days after chemotherapy, or on hormonal therapy

Dose Escalation Dose Escalation

• This trial is a dose­ranging study of four doses of the mammaglobin­A DNA vaccine

• Four groups of at least three patients will be vaccinated with mammaglobin­A DNA delivered intramuscularly at four different dose levels (150 μg, 500 μg, 1500 μg, 5 mg) every three weeks for four injections

Dose Escalation Dose Escalation

• Dose escalation will only occur when the final patient at the prior dose level has safely completed all four injections and no dose­limiting toxicity (DLT) has been noted in more than one patient at the final post­ vaccination visit

Immune Monitoring Immune Monitoring

• ELISPOT assays, intracellular cytokine expression analyses using multi­ parameter flow cytometry, and peptide MHC tetramer analyses will be used to assess the antigen­specific T­cell response to the mammaglobin­A DNA vaccine

Anticipated Toxicity Anticipated Toxicity

• Based on experience with DNA vaccines in phase I clinical trials we expect toxicity to be limited to grade 1 vaccine site reactions

• Other reactions that have been described include hyperglycemia, hypoalbuminemia, myalgia, chills, allergic rhinitis, cough, headache and pruritis

Safety Safety

• Safety defined as the absence of sever toxicity (grade 3 or greater) using the National Institutes of Health Common Toxicity Criteria

Optimal Dose Optimal Dose

• The dose of mammaglobin­A DNA vaccine that is associated with the maximum immune response will be considered optimal

• If there is no clear difference in the immune response to two or more doses of mammaglobin­A DNA vaccine, the lowest dose of mammaglobin­A DNA vaccine that is associated with the maximum immune response will be considered optimal

pING pING­ ­mammaglobin mammaglobin­ ­A vector A vector

pING­Mammaglobin 4851 bp

Ori Kanamycin

Promoter Mammaglobin

Exon

EcoRI (3324)

NheI (2919)

pING­Mammaglobin 4851 bp

Ori

Kanamyc in

P romote r

M ammaglobin

Ex on

EcoRI (3324 )

NheI (2919 )

AatI (3669 )

Acc III (244 1)

AflII (2675)

Alw 44I (584)

Apa I (3373)

ApaLI (584 )

BclI (3387)

BfrI (2675)

BglII (1093)

Bpu1102I (2162)

Bsa I (2900)

BseAI (2441)

Bsp 120I (3369 )

BspEI (2441)

BsrBI (3356 )

Bsr FI (4335)

BsrGI (1285)

BstXI (3346)

Bsu 36 I (92)

CelII (2162)

Cfr10I (4335)

DraIII (4827)

DrdI (796)

EcoNI (4380)

EcoRV (3336)

Esp I (2162)

HindIII (4171)

HpaI (2760)

KspI (1997)

Mlu I (2881)

MroI (2441)

NdeI (1576)

Not I (3351)

NruI (4636)

PmeI (3378)

PpuMI (2333)

Pvu I (4295)

PvuII (2706)

Sa c II (1997)

Sap I (2459 )

Sca I (2790)

Sma I (4419 )

SnaBI (1682)

Sno I (584 )

Spe I (134 1)

Sph I (2097)

Ssp BI (1285)

Stu I (3669)

XbaI (3363)

XcmI (2205)

XmaI (4417)

Asp 700 (3083)

Asp 700 (3143)

BanI (1807)

BanI (3465)

BpmI (1955)

BpmI (2488)

BsaAI (1682)

Bsa AI (2436)

Bsm I (4334)

Bsm I (4411)

BspDI (1173)

BspDI (4600)

BspHI (178)

BspHI (4771)

BssHII (2804 )

BssHII (2806)