Transcript No Slide Title

Membrane-bound antibodies for
therapy and imaging
Steve Roffler
Institute of Biomedical Sciences
Academia Sinica
Taipei, Taiwan
Surface expression of biologically-active proteins
enzyme
cytokine
antibody
Localized cytokine expression
Immune cell regulation
Gene expression imaging
LS
TM
Prodrug Activation
Metabolic regulation
Single-chain antibody receptors
scFv
Liao et al., Biotechnology & Bioengineering (2001) 73, 313-323
Making a scFv from a hybridoma
Gilliland et al., Tissue Antigens. 1996 47:1-20
Making a scFV from a hybridoma II
http://www.ibms.sinica.edu.tw/~sroff/protocols/scFv.htm
Expression of scFv with commercial pHook-1 vector
pcDNA3
2 C 1 1-P D G F R
Enhancing surface expression
transmembrane domains
Summary of AFP-TM results
Epitope tag
Epitope tag
m yc TM
AFP
LS HA
Sal I
Sfi I
Import to ER
B7-1
CD80
DAF
decayaccelerating
factor
ASGPR
asialoglycoprotein
receptor
cyt
Protein of interest
t1/2
Anchor in
plasma membrane
B7-1
DAF
ASGPR
PDGFR
12.2
3.8
2.4
1.6 h
AFP-B7 is most rapidly transported to the cell surface
PDGFR
platelet-derived
growth factor
receptor
AFP-B7 is more stable on the cell surface
Surface expression: B7 > DAF >> PDGFR = ASGPR
B7 TM is good for surface expression
Effect of linker domains on scFv receptor expression
Linker domains increase
surface expression
Liao et al., Cancer Gene Therapy (2003) 10: 779-790
Glycosylation controls surface shedding
IP of culture medium
protein
expression
surface
expression
GPI anchor
glycosylated
no
glycosylation
Carbohydrate chains reduce shedding
and enhance surface expression
Activation of T cells by surface anti-CD3 scFv
Defects or down-regulation
proteosome subunits
TAP-1
TAP-2
Beta 2-microglobulin
MHC class I heavy chain
Early events in T-cell activation
Direct CD3 ligation can initiate T cell activation
T cells can bind to cells that express anti-CD3 scFv
Anti-CD3 scFv activity
anti-CD3 scFv can
induce CTL activity
anti-CD3 scFv with CD86
stimulates IL-2 secretion
no stimulator cells
anti-phOx
60000
IL-2 concentration (pg/mL)
.
anti-CD3
anti-CD3 / CD86
40000
20000
Cytotoxicity (% control)
.
100
75
50
25
0
0
1
2
3
4
5
Day
Anti-CD3 scFv is active in vitro
Tumor size (mm3)
Tumor sizes in mice injected with B16-F1 transfectants
2000
0
10
20
30
phOx (control scFv)
0/6
1000
0/6
1000
0
2000
B16-F1
40
0
0
10
Tumor size (mm3)
2000
phOx + CD86
0
0
0/5
10
20
Days
30
40
Days
Days
1000
20
30
2000
1000
40
0
0
2000
aCD3
0/7
10
1000
20
Days
30
40
0
0
aCD3 + CD86
6/13
10
20
30
40
Days
anti-CD3 scFv with CD86 prevented growth
of poorly immunogenic tumors in 45% of mice
Development of systemic anti-tumor immunity in tumor-free mice
Original tumor
B16/F1 rechallenge
(tumor-free/total)
naive
0/4
B16/aCD3 + CD86
4/4
Long-term protective immunity was
established by anti-CD3 and CD86
anti-CD28 scFv receptor
scFv (aCD28)
Linker(eB7)
TM (B7)
Can bind CD28 but not CTLA-4
Is anti-CD28 scFv better than CD86?
Tumor sizes after adenoviral therapy
control
anti-phOx
2000
anti-phOx
+
CD86
2000
1500
1500
1500
1000
1000
1000
1000
500
500
500
500
0
0
10
20
0
30
0
10
Days
20
30
0
0
0
10
Days
2000
20
30
0
10
Days
anti-CD3
2000
20
30
Days
2000
anti-CD3
+
CD86
1500
1500
1000
1000
1000
500
500
500
0
anti-phOx
+
anti-CD28
2000
1500
Tumor size (mm3 )
Tumor size (mm3 )
2000
anti-CD3
+
anti-CD28
1500
1/7
0
10
20
Days
30
0
0
10
20
Days
30
0
0
10
20
30
Days
anti-CD3 with anti-CD28 delayed tumor growth
Goal:
Develop membrane-anchored chimeric proteins
that can be employed for both
gene-expression imaging and therapy
Single-gene for imaging and therapy
Reporter genes
Exogenous genes
green fluorescent protein
luciferase
herpes simplex type 1 virus thymidine kinase
cytosine deaminase
–galactosidase
High selectivity, but immunogenic
Endogenous genes
dopamine D2 receptor
transferrin receptor
Low immunogenicity, but less selective
Antibody-hapten imaging of transgene expression
Hydrophilic
probe
anti-hapten
scFv
Advantages of antibody/hapten system
Low immunogenicity
(human or humanized Ab)
Cell
High specificity and affinity
Hydrophilic probes
(small volume of distribution)
DNS probes for gamma camera imaging
C
N
O
O
phOx
dansyl
Roffler et al., Gene Ther., 13:412-20, 2006
In vivo accumulation of radioactive DNS-probe
% injected dose /g tissue
10
4h
24 h
48 h
1
0.1
0.01
Blood
DNS scFv tumor
The DNS probe was retained at tumors
that express DNS scFv on their surface
In vivo imaging
anti-DNS
tumor
anti-phOX
tumor
Mouse 2
Mouse 1
#1
1h
#2
#1
24 h
#2
#1
48 h
Dansyl probe allowed imaging of
DNS-positive tumors in mice
#2
Hapten-directed therapy
Glucuronide prodrug HAMG
COOH
O
HO
O
HO
OH
N
Active drug pHAM
Cl
HO
Cl
Cl
N
Cl
DNS-PEG-IL-2
DNS-PEG-G
DNS
DNS
anti-DNS
scFv
Prodrug: Reduce tumor size/generate antigens
IL-2: Stimulate antitumor immunity
Binding of hapten-modified proteins to anti-DNS scFv on cells
DNS-labeled proteins selectively bound
to anti-DNS scFv on CT-26 cells
Activity of DNS-PEG-IL-2
DNS-PEG-IL-2 is active when retained
by anti-DNS scFv on CT-26 cells
Prodrug activation by DNS-PEG-G
Glucuronide prodrug HAMG
Active drug pHAM
COOH
HO
HO
O
Cl
O
OH
N
Cl
Cl
HO
N
Cl
DNS-PEG-G can activate HAMG at CT-26/DNS cells
In vivo localization of DNS-PEG-G at CT-26/DNS tumors
DNS-labeled G can accumulate at CT-26/DNS tumors
Combination therapy of CT-26/DNS tumors
None of the treatments delayed the growth of CT-26/phOx tumors
Combined treatment was more effective
than single agent therapy
Chuang et al., Bioconjugate Chem., 17: 707-714, 2006.
Institute of Biomedical Sciences
Academia Sinica
Surface expression
Tang-Bi Liu
Chien-I Su
Jill Lin
T cell activation
Shih-en Chang
Kaohsiung Medical
University
Chin-Chuan Chen
Yi-Hsuan Chiang
Joseph Lee
Surface scFv
Dr. Tian-Lu Cheng
Dr. Hsin-Ell Wang
National Yang Ming
University
Dr. Yu-Ling Leu
Chia-Nan College of
Pharmacy and Science
Dr. Kuang-Wen Liao
Bing-Mae Chen
Dr. Ji-Wang Chern
National Taiwan
University