Membrane-bound antibodies for therapy and imaging
Steve Roffler
Institute of Biomedical SciencesAcademia SinicaTaipei, Taiwan
Immune cell regulation
Prodrug Activation
Gene expression imaging
Localized cytokine expression
Surface expression of biologically-active proteins
Metabolic regulation
cytokineantibody
enzyme
TMLS
TMLS scFv
VL VH
VH VL
VH
VL
CH1
CH2
H
CH3
CL
scFv
Single-chain antibody receptors
Liao et al., Biotechnology & Bioengineering (2001) 73, 313-323
Hybridoma cells
Extract RNA RT-PCR3' 5'
1st strand cDNA
antisense primer100 bp downstreamof J -C junction terminal
transferase
dGTP
3' 5'-GGGGGGGGGGGGG3' 5'GGGGGGGGGGGGGCCCCCCCCCCCCC 3'
extend5'
PCR amplify
3' 5'GGGGGGGGGGGGGCCCCCCCCCCCCC 3'5'
LS VL CK
LS VH CH1
Gilliland et al., Tissue Antigens. 1996 47:1-20
Making a scFv from a hybridoma
VL VH
PCR
subclone into pBluntsequence
VL VH
assembly PCR
VL VH
VL VH
http://www.ibms.sinica.edu.tw/~sroff/protocols/scFv.htm
Making a scFV from a hybridoma II
N N
N C N
C N C C
Type-I Type-II GPI-anchor
Dimer
GPI
1 domain
plasma membrane
hinge
CH2
CH3extracellular
intracellular
Enhancing surface expression transmembrane domains
1/2
12.2 3.8 2.4 1.6 h
B7-1 DAF ASGPR PDGFR
AFP-B7 is more stable on the cell surface
Surface expression: B7 > DAF >> PDGFR = ASGPR
Summary of AFP-TM results
AFP-B7 is most rapidly transported to the cell surface
Sfi I Sal I
LS HA AFP myc TM cyt
Import to ER Protein of interest Anchor inplasma membrane
Epitope tag Epitope tag
B7 TM is good for surface expression
B7-1 CD80
DAF decay- accelerating factor
ASGPR asialoglycoproteinreceptor
PDGFR platelet-derivedgrowth factorreceptor
.
2C11-1-B72C11-B7
2C11-e-B7
101 102 103 104
2C11-BGP-B7
2C11-CD44-B7
101 102 103 104
2C11-mBGP-B7
Fluorescent intensity
Rela
tive c
ell
num
ber
2C11-BGP-B7
2C11-e-B7
2C11-1-B7
2C11-CD44-B7
2C11-B7
2C11-mBGP-B7
HA
scFv B7
Linker domains increase surface expression
Effect of linker domains on scFv receptor expression
Liao et al., Cancer Gene Therapy (2003) 10: 779-790
IP of culture medium
Glycosylation controls surface shedding
Carbohydrate chains reduce sheddingand enhance surface expression
proteinexpression
surfaceexpression
GPI anchor glycosylatedno
glycosylation
SS
tumor cell
T cell
activation
CD3TCR
Activation of T cells by surface anti-CD3 scFv
Defects or down-regulation
TAP-1
TAP-2Beta 2-microglobulin
MHC class I heavy chain
proteosome subunits
.
Anti-CD3 scFv activity
anti-CD3 scFv can induce CTL activity
anti-CD3 scFv with CD86 stimulates IL-2 secretion
0
20000
40000
60000
Day1 2 3 4 5
anti-phOx
anti-CD3
anti-CD3 / CD86
IL-2
co
nce
ntr
atio
n (
pg
/mL
)
no stimulator cells.
0
25
50
75
100
Cyt
oto
xici
ty (
% c
on
tro
l)
anti-
CD3
anti-
CD3 +
CD86
anti-
phOx
anti-
phOx +
CD86
Anti-CD3 scFv is active in vitro
B16-F1
0 10 20 30 400
1000
2000
Days
Tum
or
size
(m
m3)
0/6
CD3 + CD86
0 10 20 30 400
1000
2000
Days
6/13
CD3
0 10 20 30 400
1000
2000
Days
0/7
phOx (control scFv)
0 10 20 30 400
1000
2000
Days
0/6
phOx + CD86
0 10 20 30 400
1000
2000
Days
Tum
or
size
(m
m3)
0/5
anti-CD3 scFv with CD86 prevented growth of poorly immunogenic tumors in 45% of mice
Tumor sizes in mice injected with B16-F1 transfectants
Development of systemic anti-tumor immunity in tumor-free mice
Long-term protective immunity wasestablished by anti-CD3 and CD86
Original tumor
B16/F1 rechallenge
(tumor-free/total)
B16/CD3 + CD86 4/4
naive 0/4
anti-CD28 scFv receptor
scFv (CD28)
TM (B7)
Linker(eB7)
Is anti-CD28 scFv better than CD86?
Can bind CD28 but not CTLA-4
Tumor sizes after adenoviral therapy
anti-CD3 with anti-CD28 delayed tumor growth
0 10 20 300
500
1000
1500
2000
Days
Tu
mor
siz
e (
mm3)
0 10 20 300
500
1000
1500
2000
Days
0 10 20 300
500
1000
1500
2000
Days
0 10 20 300
500
1000
1500
2000
Days
0 10 20 300
500
1000
1500
2000
Days
Tu
mor
siz
e (
mm3)
0 10 20 300
500
1000
1500
2000
Days
0 10 20 300
500
1000
1500
2000
Days
control anti-phOx
anti-CD3+
anti-CD28
anti-phOx+
anti-CD28
anti-phOx +
CD86
anti-CD3+
CD86
anti-CD3
1/7
Goal:
Develop membrane-anchored chimeric proteins that can be employed for both
gene-expression imaging and therapy
Single-gene for imaging and therapy
green fluorescent proteinluciferaseherpes simplex type 1 virus thymidine kinasecytosine deaminase –galactosidase
dopamine D2 receptortransferrin receptor
High selectivity, but immunogenic
Low immunogenicity, but less selective
Reporter genes
Exogenous genes
Endogenous genes
Antibody-hapten imaging of transgene expression
Advantages of antibody/hapten system
Low immunogenicity (human or humanized Ab)
High specificity and affinity
Hydrophilic probes (small volume of distribution)
anti-haptenscFv
Cell
Hydrophilic probe
DNS probes for gamma camera imaging
dansylphOx
O
C
O
N
N
CH3CH3
SO2 NH C
O
CH2
C
O
CH2HO
N (CH2)2 N
CH2 C OH
O
(CH2)2 N
CH2
CH2
C
O
C
O
OH
NH2
N
CH3CH3
SO2 NH C
O
CH2
C
O
CH2HO
N (CH2)2 N
CH2 C OH
O
(CH2)2 N
CH2
CH2
C
O
C
O
OH
NH SO2
N
CH3CH3
Roffler et al., Gene Ther., 13:412-20, 2006
Blood DNS scFv tumor0.01
0.1
1
10 4 h
24 h
48 h
% i
nje
cted
do
se /
g t
issu
e
In vivo accumulation of radioactive DNS-probe
The DNS probe was retained at tumorsthat express DNS scFv on their surface
In vivo imaging
anti-phOX tumor
Mouse 1 Mouse 2
#1 #2
anti-DNS tumor
1 h 24 h 48 h#1 #2 #1 #2
Dansyl probe allowed imaging ofDNS-positive tumors in mice
DNS
DNS-PEG-IL-2
DNS
DNS-PEG-G
OHOHO
OH
COOH
O NCl
Cl
Glucuronide prodrug HAMG
NCl
ClHO
Active drug pHAM
Hapten-directed therapy
anti-DNSscFv
Prodrug: Reduce tumor size/generate antigensIL-2: Stimulate antitumor immunity
Binding of hapten-modified proteins to anti-DNS scFv on cells
DNS-labeled proteins selectively boundto anti-DNS scFv on CT-26 cells
OHO
HOOH
COOH
O NCl
Cl
Glucuronide prodrug HAMG
N
Cl
ClHO
Active drug pHAM
Prodrug activation by DNS-PEG-G
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
None of the treatments delayed the growth of CT-26/phOx tumors
Combination therapy of CT-26/DNS tumors
Combined treatment was more effective than single agent therapy
Chuang et al., Bioconjugate Chem., 17: 707-714, 2006.
Dr. Kuang-Wen Liao
Tang-Bi Liu
Surface scFv
T cell activationSurface expression
Shih-en Chang
Bing-Mae Chen
Dr. Yu-Ling Leu Chia-Nan College of
Pharmacy and Science
Dr. Ji-Wang Chern National Taiwan
University
Dr. Tian-Lu ChengKaohsiung Medical
University
Chin-Chuan Chen
Dr. Hsin-Ell WangNational Yang Ming
University
Yi-Hsuan Chiang
Chien-I Su
Joseph Lee
Jill Lin