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)