Use of Therapeutic DNA
Vaccine to Control Cancer
Ming-Derg Lai, Ph.D.
Department of Biochemistry and Molecular Biology
College of Medicine
National Cheng Kung University
What is gene therapy?
Induction of antitumor immunity with combination of
HER2/neu DNA vaccine and interleukin 2 genemodified tumor vaccine.
Clin. Cancer Res. 2000 Nov;6(11):4381-8
Gene transfer
• Delivery
- Viral
• Retrovirus
• Adenovirus
• Adeno-associated virus (AAV)
• Herpes virus
- Nonviral
• Lipofection
• Direct injection of DNA (Naked DNA vaccine)
DNA Vaccine
• A DNA Vaccine is essentially a DNA sequence that
can be used as a vaccine
• This sequence of DNA comes from a piece of
pathogen or tumor antigen.
• The sequence is then injected into a cell of a person,
the protein is produced, and an immunity is created
against the infectious organism or tumor
Feature of DNA vaccine
(2)
(5)
(1)
(4)
(3)
Development of the Therapeutic
HER2/Neu DNA Vaccine to Inhibit
Mouse Tumor Naturally
Overexpressing Endogenous Neu
HER-2/neu
HER-2/neu /Oncogene
• HER-2/neu is overexpressed in many
human cancers (Ovarian, bladder, breast,
lung..)
• Overexpression of HER-2/neu is
correlated with the aggressiveness and
poor prognosis of cancer
Prevention HER-2/neu DNA vaccine
HER-2/neu-expressing Tumor model
Mouse tumor cells natively overexpressing mouse
HER-2/neu
HER-2/neu-expressing tumor model
<20 %
Fusion with activating gene
Activating gene
Fusion with activating gene
Kaplan-Meier analysis of mouse survival
100
saline(n=37)
pRc/CMV(n=16)
80
N'-neu-IL-4(n=28)
N'-neu,(n=37)*
60
N'-neu-GM-CSF(n=27)*
N'-neu-IL-2(n=37)**
40
20
0
0
10
20
30
40
50
60
70
80
90 100
HER-2/neu specific antibody
HER-2/neu
protein
10Antibody
(serum)
20Antibody
ELISA
HER-2/neu specific Cytotoxic T lymphocytes
Cytotoxic responses of spleen cells
100
90
pRc/CMV-N'-neu-IL-2
% specific lysis
80
pRc/CMV-N'-neu
70
60
pRc/CMV-N'-neu-GM-CSF
50
40
pRc/CMV
30
20
Saline
10
pRc/CMV-N'-neu-IL-4
0
50
25
effector : target ratio
12.5
Test-Negative
Specific lysis :
Total-Negative
%
Summary I
• Neu DNA vaccine is effective in controlling
tumor progression.
• Fusion of IL-2 and neu produced strongest
anti-tumor effects.
發展Neu DNA疫苗於癌症治療
(新低壓基因槍之研發)
主持人：賴明德教授 (Ming-Derg Lai)
執行人：林季千博士 (Chi-Chen Lin)
國立成功大學醫學院
生物化學暨分子生物研究所
研究計劃
• 背景：本計劃是發展利用Neu DNA疫苗治療癌症
之相關作用機轉及傳送DNA 之技術。利用發展癌
症治療性疫苗(therapeutic DNA vaccine)，我們和
生物鎵公司(Bioware Inc.)合作發展實驗室用及醫
療用基因槍(gene gun). 目前商業用基因槍價格昂
貴且產生極大噪音。
• 目的：發展低壓基因槍供實驗室及醫院使用。
• 重要性：台灣自製具[低噪音][低氦量]之多功能基
因槍，具有龐大商機。
低壓基因槍
Sample
Loading
• 此次的實驗更不同與
以往所使用之高壓式
傳統基因槍 (400 psi，
氦氣，Bio-Rad)， 我
們所使用的是經由改
良過之低壓式基因槍
(40-50 psi，氦氣，
Bio-Ware)。 此基因槍
可明顯降低噪音。(US
Patent: 6436709 B1).
主要成果
• 證明台灣製基因槍和目前商業用基因槍具
有相同治療效果。
• 證明台灣製基因槍具有投遞其他分子如裸
露DNA之能力。
測試比較基因槍療效
1 X 106 MBT-2 腫瘤細胞 in 6-8 週 C3H/HeNCrJ 老鼠
天
10
17
24
腫瘤生長
及老鼠存活
施打neu DNA
Bioware:
1. Control mice
2. One mg gold coated with 1 mg HER-2/neu plasmid DNA
Commercial high-pressure gene gun
1. Control mice
2. One mg gold coated with 1 mg HER-2/neu plasmid DNA
新式低壓基因槍(Bio-ware)與傳統高壓基因槍
(Commercial) 具相同癌症療效
老鼠存活率相同!
新式低壓基因槍(Bio-ware)與傳統高壓基因槍(Commercial)
之抗腫瘤能力之評估.
觀察老鼠之存活率.*代表比較於控制組別(只施打saline)P<0.05.
新式低壓基因槍(Bio-ware)與傳統高壓基因槍
(Commercial) 引發相同免疫反應
產生
相同量
抗体
相似
細胞毒殺
反應
低壓及高壓基因槍引發(B) 相同強度抗體反應及(C)細胞型反應
低壓新基因槍多功能特性
• 我們此次所使用之新式基因槍，具有之另
一個特性及是其可在不須經由金粒子攜帶
之情況下，直接將裸露之DNA疫苗投遞之
細胞體內，而這樣的特性剛好可以克服金
粉所帶來之免疫偏差(Th1/Th2)。
低壓新基因槍可傳送裸露DNA 入老
鼠體內表現
比較基因槍遞送金粒子coated DNA疫苗和裸露之DNA 疫苗表現量之差異.
在打完質體後第三天,分析老鼠表皮層之pCMV-luciferase 表現,
而我們是藉由分析均質化後之表皮層內Luciferase 之活性作為比較表現量之依據
當使用低壓新基因槍投遞無金粉之裸露DNA產生相
同治療腫瘤效果
治
療
效
果
無
差
異
比較基因槍遞送金粒子coated DNA疫苗和裸露之DNA 疫苗之抗腫瘤能力.
觀察老鼠腫瘤體積及老鼠之存活率.
當使用低壓新基因槍投遞無金粉之裸露DNA
產生Th1 免疫反應(治療癌症較佳)
分析基因槍遞送金粒子coated
DNA疫苗和裸露之DNA 疫苗之
Th1/Th2免疫反應.
在打完3劑疫苗後之一個星期取
出 大腿淋巴結細胞, 利用RTPCR 分析 IFN-g, IL-18 (Th1) 和
IL-4 , IL-10 (Th2) 細胞激素之蛋
白質表現量.
＊代表P<0.001
Th1 biased
Th2 biased
結論 II
(1) 新式低壓基因槍其具有能與傳統式高壓基因槍
一樣之投遞DNA和誘導免疫反應治療腫瘤之功
能當使用金粉包裝之DNA.
(2) 新式低壓基因槍可成功傳送無金粉包裝之DNA
進入生物體內。並產生免疫力治療癌症。新式
低壓基因槍傳遞之無金粉包裝之裸露DNA 引發
偏向細胞型免疫反應。
產業發展前景：
(1) 具專利保護
(2) 低噪音
(3) 低氦量使用
(4) 多功能使用包括金包埋DNA 及裸露DNA 或其
他分子。
A novel cancer therapy based on
modulating indoleamine 2,3-dioxygenase
in skin dendritic cells in vivo
Ming-Derg Lai
Department of Biochemistry and
Molecular Biology
College of Medicine
National Cheng Kung University
Aim
• Generation of anti-tumor immunity by in
vivo targeting dendritic cells.
• Rationale
1. Modifying dendritic cells is a promising
cancer therapy.
2. The novel gene gun appears to deliver
gene into DCs in an efficient way.
Target gene: indoleamine 2,3dioxygenase (IDO)
•
IDO
also
plays
an
L-tryptophan
important role in immune
escape in cancer. The
IDO
establishment of tolerance
L-kynuerine
may be mediated through
either localized depletion of
tryptophan or accumulation
of toxic metabolites.
3-hydroxylanthralinic acid • Overexpression of IDO
was observed in many
types of tumors and/or
tumor draining lymph node.
Hypothesis: Delivery of IDO siRNA can
generate anti-tumor immunity in vivo
S.C. Tumor Implantation
and tumor tolerance
Skin delivery of IDO siRNA
IDO+
IDO+
Gene gun
CD8+
T cell
Induction of cytotoxic immune
responses and tumor regression
DC
IDO-negative DC
Tumor-draining
Lymph node
IDO-negative Dendritic cells
migrate to Lymph node
Tumor
CD8+
T cell
IDO siRNA downregulates IDO but has no
effect on IDO2
Relative mRNA expression fold
(IDO/HPRT)
The effects of IDO siRNA on
dendritic cells in vivo
1.25
1.00
0.75
0.50
0.25
0.00
CD11c+ cells was harvested
from inguinal lymph node
Forty eight hours after vaccination.
Relateive mRNA expression fold
(IDO2/HPRT)
IDO siRNA
Scramble IDO siRNA
1.25
1.00
0.75
0.50
0.25
0.00
IDO siRNA
scramble IDO siRNA
Cancer therapeutic effect of IDO siRNA
and IDO inhibitor, 1-methyl tryptophan
1x106 MBT-2
5 mg/ml in drinking water, pH=9.9 1-MT treatment
……
Day0
Day8 Day15 Day22 Day29 Day36
1st vaccination and weekly vaccination, until mice were sacrificed
IDO siRNA or 1-Methyltryptophan
delays tumor growth
*
Saline (n=4)
Scramble IDO siRNA (n=5)
IDO siRNA (n=7)
1-MT (n=5)
3
Tumor volume (mm )
3000
2000
*
1000
0
5
10
15
Days after MBT-2 challenge
20
Skin delivery of IDO siRNA is more effective
than systemic administration of 1-MT
Percent survival
100
Saline (n=12)
Scrmble IDO siRNA(n=12)
IDO siRNA(n=10)**
1-MT(n=10) *
75
50
25
0
0
10
20
30
40
50
60
Days after MBT-2 challenge
70
Table.1 T cell infiltration in C3H mice model
CD8+ T cell
CD4+ T cell
NK cell
Neutrophil
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Saline
2±2
1±1
1±1
3±2
L-1MT
8 ± 5*
48 ± 12**
13 ± 5*
48 ± 7**
Scramble
IDO siRNA
2±1
3±2
3±1
14 ± 4
IDO siRNA
28 ± 4**
29 ± 7**
16 ± 11
52 ± 6**
Treatment
Random 5 field counted
* Compared with saline
** compared with saline and scramble
IDO siRNA enhances cytotoxic T
cells activity
Spleenic lysis(%)
30
*
saline
Scramble IDO siRNA
IDO siRNA
1-MT
*
20
10
0
50:1
25:1
12.5:1
Effector:Target cells
Can cells obtain anti-tumor immunity
by adoptive transfer of CD11C+ Cells
Day 0
Naïve mice vaccinated
with IDO siRNA.
Day 7
Second IDO siRNA
vaccination.
Day 9
Mice were sacrificed
and inguinal lymph
nodes were harvested.
Isolation of
cd11c+ cells
S.C. Tumor Implantation.
S.C. injection CD11c+
cells to tumor-bearing
mice at day 9.
Adoptive transfer CD11c+ dendritic
cells from vaccinated mice may
provide protection from cancer
Tumor volume (mm 3)
1000
IDO siRNA
Scramble IDO siRNA
750
500
250
0
5.0
7.5
10.0
12.5
Days after MBT-2 challenge
15.0
What about offsite effect of IDO siRNA?
Approach: analyze the therapeutic effect of
two other IDO siRNAs targeting different sequences.
pcDNA3.1-IDO
pshU6 vector
＋
＋
＋
－
＋
－
＋
－
＋
－
IDO siRNA
IDO siRNA-2
－
－
＋
－
－
＋
－
－
－
－
IDO siRNA-3
－
－
－
＋
－
Scramble IDO siRNA
－
－
－
－
＋
myc
β-actin
The therapeutic effects of IDO siRNAs are
correlated with their suppression effects
*
Tumor size (mm3)
3500
Scramble IDO siRNA (n=4)
IDO siRNA (n=3) *
IDO siRNA-2 (n=3) *
IDO siRNA-3 (n=5)
3000
2500
*
2000
*
1500
1000
500
0
5
10
15
20
25
Days after MBT-2 challenge
It suggests that the therapeutic effect of IDO siRNA
Is NOT due to offsite effect.
IDO siRNA exert anti-cancer
therapeutic effect on CT-26 tumor
cells in BALC/c mice model
Tumor volume (mm 3)
2000
*
1000
*
Saline (n=5)
Scramble IDO siRNA (n=5)
IDO siRNA (n=5) *
L-1MT (n=5)
*
0
5
15
25
Days after CT-26 challenge
35
IDO siRNA is also more effective
on CT-26 colon cancer cells
Percent survival
100
Saline (n=9)
Scramble IDO siRNA (n=9)
IDO siRNA (n=11)*
L-1MT (n=6)
75
50
25
0
0
10
20
30
40
50
60
Days after CT-26 challenge
70
Table 2. T cell infiltration in BALB/c mice model
Random 3 field counted
* Compared with saline
** Compared with saline and scramble
Whether IDO siRNA can function as
therapeutic adjuvant for DNA vaccine?
pcDNA3.1-IDO
＋
＋
＋
－
Human-cyto-N’neu
＋
－
－
－
Human-cyto-N’neu-IDO siRNA
－
＋
－
－
Human-cyto-N’neu-scramble IDO siRNA
－
－
＋
－
CMV
promoter
Neu
Neu IDO siRNA
Neu scramble
Neu DNA vaccine plus IDO siRNA
delay tumor growth most efficiently
Tumor size(mm3)
3500
saline (n=5)
IDO siRNA (n=5)
Neu (n=6)
Neu-IDO siRNA (n=7)
Neu-scramble IDO siRNA (n=5)
3000
2500
2000
1500
1000
500
IDO siRNA
0
5
10
15
20
Days after MBT2 challenge
25
CMV Neu
CMV
Neu
IDO siRNA
CMV
Neu
scramble
IDO siRNA acts as adjuvant to enhance
therapeutic efficacy of neu DNA vaccine
Percent survival
100
Saline (n=9)
neu (n=14)*
neu-IDO siRNA (n=16)**
IDO siRNA (n=12)*
Neu-scramble IDO siRNA (n=5)
50
0
0
30
60
Days after MBT-2 challenge
90
Summary III
• Skin delivery of IDO siRNA can exert cancer
therapeutic effect in two different mouse tumor
animal models.
• Advantages and applications:
1. Simple form of drug: No preparation of DCs for
ex vivo loading.
2. It can function alone and had even stronger anticancer therapy in combination with other DNA
vaccines.
3. The IDO siRNA can be replaced with other
immunoregulatory genes in DCs.
4. This provides an alternative method to analyze
the gene function in skin dendritic cells in vivo,
as shown by IDO 1 and IDO2.
TSP-1 (Thrombospondin-1)
• TSP-1 was first isolated from platelets that
had been stimulated with thrombin.
• TSP-1 has multiple receptors, among
which CD36, CD47 and integrins are of
particular note.
The structure of TSP-1
Matrix structure
Collagen
Fibronectin
Laminin
proteoglycans
Extracellular proteases
MMPs
plasmin
Inactive TGF-β
active TGF-β
Adhesion/migration
integrins
CD47
proteoglycans
PI3-K
FAK
Ras
P38-MAPK
Extracellular signaling
TSP-1
Intracellular signaling
Cytoskeletal organization
PI3-K
Fascin
PKCa
Muskelin
Apotosis
CD36
CD47
Proteoglycans
P38-MAPK
Fyn
caspases
TSP-1 in immune system
• CD47 ligation induces a rapid caspase-independent apoptosis-like cell
death in human monocytes and dendritic cells.
Scand . J. Immunol. 2004 Jan;59(1):40-9
• Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation.
J Immunol. 2001 Feb 15;166(4):2427-36. 64
• CD47 engagement inhibits cytokine production and maturation of human
dendritic cells.
J Immunol. 2000 Feb 15;164(4):2193-9
• Thrombospondin-1 and indoleamine 2,3-dioxygenase are major targets
of extracellular ATP in human dendritic cells.
Blood. 2005 Aug 23
TSP-1 may be a immune suppressor~
Aim
+
neu DNA vaccine
TSP-1 siRNA
Deliver the DNA plasmid
Control
N’-neu-IL-4
N’-neu
into DCs by gene gun
N’-neu-IL-2
=
N’-neu-GM-CSF
Enhance the therapeutic efficacy of neu DNA vaccine?
To check the TSP-1 siRNA efficiency
To prove the knockdown effect of TSP-1
siRNA in vivo
Control
N’-neu-IL-4
N’-neu
c-myc
β-actin
N’-neu-GM-CSF
In vitro
N’-neu-IL-2
By gene gun in vivo??
U6 vector 10μg/single
micex6
TSP-1 siRNA 10μg /single
micex6
２4ｈｒ
２4ｈｒ
sacrifice
sacrifice
Lymph node
Lymph node
Culture O/N
Western blotting
Collect supernatant
Gene gun could deliver TSP-1 siRNA into dendritic
cells in draining lymph node
Human-cyto-N’-neu-TSP-1
siRNA construction
By CCLin
To check the function of human-cyto-N’-neu-TSP-1
siRNA efficiency
The therapeutic effect of human-cyto-N’-neu-TSP-1
siRNA vaccination
Tumor volume
Survival curve
Percent survival
125
saline(n=11)
TSP-1siRNA (n=4)
human-cyto-N'-'neu (n=15)*
human-cyto-N'-neu-TSP-1siRNA (n=19)***
100
75
50
25
0
0
10
20
30
Time
40
50
Summary IV
• Similar approach may be applied to other
negative immune regulatory molecules,
such as TSP-1.
Acknowledgements
•
•
•
•
Meng-Chi Yen (顏孟畿)
Chi-Chen Lin (林季千)
Shih-Shien Huang (黃仕憲)
Yi-Ling Chen (陳怡玲)
• Professor Huan-Yao Lei (黎煥耀)
• Chih-Peng Chang (張志鵬)
EGFR DNA vaccine on Lewis lung
carcinoma cells in mice
1x106 LL2
0
5
12
19
Sec-N’-EGFR DNA vaccine
Tumor sizes(mm3)
Day
2000
1800
1600
1400
1200
1000
800
600
400
200
0
Control
Gene Gun
Intramuscular
Gene
Gun+Intramuscular
5
8
11
14
17
Percent survival
100
Control(n=7)
I.M(n=6)*
G.G(n=7)*
G.G+I.M(n=6)**
75
50
25
0
0
10
20
30
40
50
Days
Mice were killed when Tumor volume exceeded 2000mm3 or the mouse
was in poor condition and expected shortly to become moribund