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Antibody and prodrug therapy of cancer
Steve Roffler
Institute of Biomedical Sciences
Academia Sinica
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Overview Cancer drug therapeutic index
Antibody targeted prodrug activation
9ACG - new glucuronide prodrug
Surface expression of proteins
scFv activation of T cells
Membrane suicide gene therapy
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Effective medicines require a therapeutic index > 1
Toxic drug concentrationTherapeutic concentration
Property of tumor cells
Unique or over-expressed target in cancer cells
Tumor targeting
Relative resistance of normal cells
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Alkylating agents kill dividing cells
X NCH2
ClCH2 CH2
ClCH2X N
CH2
CH2CH2
ClCH2+
X NCH2
CH2 CH2
ClCH2
RN
H
aziridinium intermediate
R N H
H
+
NN
NN
O HH
H2N
dRP
N-7 position of guanine
NN
NN
O
H
H2N
dRP
X NCH2
CH2CH2
ClCH2
A G T C T
T C A G A
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Unique or over-expressed target in cancer cells
Property of tumor cells
Tumor targeting
Relative resistance of normal cells
Basis for cancer drug therapeutic index
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Gleevec
Nature Reviews Drug Discovery 1: 493-502, 2002
Inhibition ofautophosphorylation
IC50 (µM)
v-ABL 0.1-0.3
BCR-ABL 0.25
PDGF receptor 0.1
c-KIT 0.1
FLT3 >10
c-FMS >10
EGF receptor >100
c-ERBB2 >100
insulin receptor >100
IGF-1 receptor >100
JAK2 >100
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.
0.1
1
10
Normal cervix Cervical cancer
Top
o I
acti
vity
(h
-1)
Topoisomerase I inhibitors
O
O
NN
N
N
O
CH2
CH3
HOC2H5
O
O
CPT-11 (Irinotecan)
HO
N
N
O
CH2
CH3
HOC2H5
O
O
SN-38
carboxyesterase
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Unique or over-expressed target in cancer cells
Property of tumor cells
Tumor targeting
Relative resistance of normal cells
Basis for cancer drug therapeutic index
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Cyclophosphamide metabolism
ClCH2N
ClCH2
P
ON
H
CH2
CH2 OH2C
CH2
CH2
CH
OH
ClCH2N
ClCH2
P
ON
H
CH2 OH2C
CH2
CH2
ClCH2N
ClCH2P
ON
H
CH2 O
CH2
CH2CH2
H
C
O
H
ClCH2N
ClCH2P
ON
H
O
CH2
CH2
H
ClCH2N
ClCH2P
ON
H
CH2 O
CH2
CH2CH2
H
C
O
HO
CH2CHC
O
H
microsomal oxidation (liver)
Cyclophosphamide
4-Hydroxycyclophosphamide
Aldophosphamide
Aldehyde dehydrogenase
Carboxyphosphamide
Phosphoramide mustard
Acrolein
+
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Unique or over-expressed target in cancer cells
Property of tumor cells
Tumor targeting
Relative resistance of normal cells
Basis for cancer drug therapeutic index
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Nature Cancer Reviews 1: 118-129, 2001
Antibody-targeted cancer therapy
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Nature Cancer Reviews 1: 118-129, 2001
Antibody name
Target Tumortarget
Status
Panorex EpCam Dukes' C CRC Approved inGermany 1995
Rituxan CD20 NHL Approved 1997
Herceptin ERBB2 Metastaticbreast cancer
Approved 1998
Mylotarg CD33 AML Approved 2000
Campath CD52 B-cell CLL Approved 2001
Zevalin CD20 NHL Approved 2002
Theragyn PEM ovariangastric cancers
Phase III
Avastin VEGF Metastatic NSCLC Phase III
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Antibody-directed enzyme prodrug therapy
21
Tumor cell
Prodrug
Active
Enzyme
Tumor cell
Drug
mAb
∆ Amplification step ∆ Bystander killing∆ Flexibility
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ADEPT
OHO
HOOH
COOH
O NCl
Cl
Carcinoma
Glucuronide prodrug
NCl
ClHO
Active drug
Immunoenzyme
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Summary of previous findings
BHAMG 100-1000 times less toxic than pHAM in vitro
Cured rat hepatocellular carcinoma tumors
Produces strong bystander effect in vitro and in vivo
Mechanism of low prodrug toxicity
Clearance of immunoenzyme improves tumor localization
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Immunoenzymes for human cancer therapy
+ mAb B72.3 against TAG-72
+ on colon, breast, ovarian, lung cancers
+ employed for imaging in over 1000 patients
+ TAG-72 in not internalized
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Immunoenzyme localization
Tumor
Blood
Lung
Liver
Spleen
Kidney
Intestine
Colon
125I-B72.3-ßG-PEG
1
28.5
33.3
18.9
26.3
30.7
120
39.7
Tumor/TissueTissueG
125I ***
Remove blood, organs after 24 h
Measure cpm/mg
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ADEPT therapy of human colon xenografts
G
OHO
HOOH
COOH
O NCl
Cl
.
0
500
1000
1500
2000
2500
10 20 30 40 50 60 70
Day
PBS
BHAMG
pHAM
H25-G-PEG
B72.3-G-PEG
Me
an
tu
mo
r si
ze (
mm
3 )
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ADEPT therapy of rat malignant ascites
Inject BHAMG prodrug
Day 0 Inject 1.5x107 ADS-30D cells
Day 1 Inject 1.5x107 AS-30D cells
Day 7 Inject RH1-G immunoenzyme
Experiment Non-treated ADEPT
1 0/6 19/20
2 0/6 15/163 0/4 12/12
4 0/4 11/115 0/6 21/22
6 0/6 24/247 0/4 7/88 0/4 15/169 0/6 30/30
10 0/2 2/2
Total 0/48 156/161
Cures/Total
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In vivo depletion of immune cells during ADEPT
.
0
50
100
0 25 50 75 100
Days
ADEPT
notreat CD4-/CD8-
CD8-
CD4-
NK- M-
Su
rviv
al (
%)
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T cells are important for control of metastasis during ADEPT
non-depleted T cell depleted
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Glucuronide prodrug of camptothecin
- poor water solubility
- converted to inactive form in vivo
- high toxicity
N
HO O
N
O
O
OO
OH
OHHO
COO-
N
HO O
N
O
O
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OOH
HOOH
CO
OH
O
N
N
O
O
HO O
O
O
NH
9-ACG
G
N
N
O
O
HO O
NH
O+
-CO2
9-AC
9ACG 9-aminocamptothecin glucuronide
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Properties of 9ACG
- 100 to 2000 times more soluble than 9AC
- stable in human serum for >72 h
Km = 31.0 µM Vmax = 10.9 µmole/µg-min
.
0
25
50
75
100
125
0.1 1 10 100 10009ACG + ßG
9ACG
9AC
topotecan
Concentration (nM)
DN
A s
yn
the
sis
(%
co
ntr
ol)
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.
1
10
100
0 50 100 150 200
HSA
PBS
1
10
100
0 50 100 150 200 250
Time (min)
9AC
9ACGL
ac
ton
e f
orm
(%
)
9ACG is not deactivated by HSA
N
N
O
OH O
O
N
N
OH
O
COOH
OH
Albumin
N
N
OH
O
COOH
OH
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9ACG activity against human lung cancer
.
0
500
1000
1500
2000
0 10 20 30 40 50
Time (days)
0
100
200
0 5 10 15 20
Tu
mo
r si
ze (
mm
3 )
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.
0
500
1000
1500
2000
10 20 30 400
500
1000
1500
2000
10 20 30 400
500
1000
1500
2000
10 20 30 40
70
80
90
100
110
10 20 30 40
Topotecan
9ACG
PBS
Tum
or
siz
e (
mm
3 )
Days
Days
Bo
dy
wei
gh
t (%
co
ntr
ol)
PBS Topotecan9ACG
Low dose 9ACG vs. topotecan
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human tumor
Mechanism of 9ACG activation
9ACG activated by ß-glucuronidase at tumor?
Source of ß-glucuronidase - tumor or immune cells?
anti-mouse ß-glucuronidase
anti-human ß-glucuronidase
ß-glucuronidase KO tumor
ß-glucuronidase KO mice