Post on 28-Jan-2016
Novel Chemotactic-Antigen DNA VaNovel Chemotactic-Antigen DNA Vaccineccine againstagainst CancerCancer新型肿瘤趋化抗原基因疫苗新型肿瘤趋化抗原基因疫苗
Novel Chemotactic-Antigen DNA VaNovel Chemotactic-Antigen DNA Vaccineccine againstagainst CancerCancer新型肿瘤趋化抗原基因疫苗新型肿瘤趋化抗原基因疫苗
Cancer Institute, HospitalCancer Institute, Hospital
Chinese Academy of Medical SciencesChinese Academy of Medical Sciences
Peking Union Medical CollagePeking Union Medical Collage
Shuren ZhangShuren Zhang
张叔人张叔人
Intratumoral SLC administration promotes Th1 cytokine and antiangiogenic
chemokine release and a decline in immunosuppressive mediators
Th1 cytokine : IFN-, GM-CSF, IL-12, MIG, and IP-10
Immunosuppressive mediators: PGE2, IL-10, and TGF-
angiogenic factor: VEGF
The Journal of Immunology, 2000, 164: 4558–4563. Steven M. Dubinett
Regulation of Cytokines Regulation of Cytokines secretionsecretion by by seconsecondary lymphoid tissue chemokine (SLC)dary lymphoid tissue chemokine (SLC)
Chemotactic-Antigen DNA Vaccine Chemotactic-Antigen DNA Vaccine ((CADV)CADV)
趋化抗原基因疫苗趋化抗原基因疫苗AgAg
Her2,TERTHer2,TERT
PSA,PAPPSA,PAP
CEA,HPVCEA,HPV
AgAg
Her2,TERTHer2,TERT
PSA,PAPPSA,PAP
CEA,HPVCEA,HPV
SLCSLCSLCSLC FcFcFcFcCMVCMVCMVCMV
Enzyme Cut SitesEnzyme Cut SitesEnzyme Cut SitesEnzyme Cut Sites
DC Th
CTL
T
B
CCR7
FcR
CCR7
Gene transfer and expression
Recruiting DC and T cell ; catching Ag by ligands; Ag pre
sentation
T and B cell activation, anti-Tumor Effects
Effect Mechanisms of Effect Mechanisms of CADVCADVEffect Mechanisms of Effect Mechanisms of CADVCADV
Tumor
CMV SLC Ag Fc
EPMHC-II
MHC-I
Injection
or
Gene gun
Antigen presentation by DCs through ligand-mediated internalization can be enhanced up to 10,000-fold over fluid-phase antigen pinocytosis
Mouse Model of CADVMouse Model of CADV
mSLCmSLC FcFcCMVCMV
Her2/P53Her2/P53HPV16E7HPV16E7
pSLC-Ag-Fc
SLC
Ag
Fc
CMV
E7
Mouse Model of CADVMouse Model of CADV
(pSLC-E7-Fc)(pSLC-E7-Fc)
Mouse Model of CADVMouse Model of CADV
(pSLC-E7-Fc)(pSLC-E7-Fc)
RT-PCR and RT-PCR and Western Blotting AnalysisWestern Blotting Analysis RT-PCR and RT-PCR and Western Blotting AnalysisWestern Blotting Analysis
(A) RT-PCR detection of B16F10 cells transfected with various recombinant plasmids. DNA marker (M),Lane 1 (vector control), Lane 2 (SLC fragment), Lane 3 (Sig-E7 fusion fragment), Lane 4 (SLC-E7 fusion fragment),Lane 5 (Sig-Fc fusion fragment), Lane 6 (SLC-Fc fusion fragment), Lane 7 (Sig-E7-Fc fusion fragment), Lane 8 (SLC-E7-Fc fusion fragment). (B) Western blotting analysis of B16F10 transfectants. SLC-E7-Fc fusion protein (50kD), E7-Fc fusion protein (38kD),SLC-E7 fusion protein (22kD).
Chemotaxis Assay Chemotaxis Assay by Chemotaxis Chamberby Chemotaxis Chamber Chemotaxis Assay Chemotaxis Assay by Chemotaxis Chamberby Chemotaxis Chamber
pSLC-E7-Fc pcDNA pSLC-E7-Fc pcDNA
HPV16-E7 as tumor antigen. E7+C3 tumor cell subcutaneous inoculated in C57BL/6 mice on day0. The tumor bearing mouse immunized by gene gun with different gene vaccine on day 6, day 13 and day 20.
Therapeutic immunization with pSLC-Therapeutic immunization with pSLC-E7-Fc induces C3 tumor regressionE7-Fc induces C3 tumor regression
Therapeutic immunization with pSLC-Therapeutic immunization with pSLC-E7-Fc induces C3 tumor regressionE7-Fc induces C3 tumor regression
0
0. 5
1
1. 5
2
2. 5
3
3. 5
4
4. 5
5 7 9 11 13 15 17 19 21 23 25 27 29 34
Days af ter tumor i nocul at i on
Tumo
r si
ze (
mm3 )
vect or
pSLC- Fc
psi g- E7
psi g- E7- Fc
pSLC- E7
pSLC- E7- Fc
Group Tumor free
pCpSLC-Fcpsig-E7
pSLC-E7psig-E7-Fc
pSLC-E7-Fc
0/8 (0%)0/8 (0%) 4/8 (50%) 3/8 (38%) 3/8 (38%) 8/8 (100%)
0
0. 5
1
1. 5
2
2. 5
14 20 24 27 30 36
Dats af ter tumor i nocul at i on
Tumo
r si
ze C
M3
pAdpAd- SLCpAd- FcpAd- SLC- FcpAd- E7pAd- SLC- E7pAd- E7- FcpAd- SLC- E7- Fc
HPV16-E7 as tumor antigen. E7+TC-1 tumor cell subcutaneous inoculated in C57BL/6 mice on day0. The tumor bearing mouse immunized by gene gun with different gene vaccine on day 15, day 22 and day 28.
Therapeutic immunization with pSLC-E7-Fc Therapeutic immunization with pSLC-E7-Fc induces TC-1 tumorregressioninduces TC-1 tumorregression
Therapeutic immunization with pSLC-E7-Fc Therapeutic immunization with pSLC-E7-Fc induces TC-1 tumorregressioninduces TC-1 tumorregression
Recharlenge with 1.8 x 105 TC-1, after 90days later of first TC-1 inoculation.
Tumor incidence after TC-1 tumor recharlengeTumor incidence after TC-1 tumor recharlengedd
Tumor incidence after TC-1 tumor recharlengeTumor incidence after TC-1 tumor recharlengedd
0
20
40
60
80
100
0 6 10 14 20 25 30 34
Days after tumor recharlenged
Tum
or in
cide
nce
(%)
NC 5/ 5
pAd- SLC- E7- Fc 0/ 5
Prostate Cancer Chemotactic-AntigeProstate Cancer Chemotactic-Antigen DNA Vaccine n DNA Vaccine
前列腺癌趋化抗原基因疫苗前列腺癌趋化抗原基因疫苗
Enzyme Cut SitesEnzyme Cut SitesEnzyme Cut SitesEnzyme Cut Sites
SLCSLC FcFcCMVCMV
Her2/P53Her2/P53PSA-mPAP-PSMPSA-mPAP-PSM
(3P)(3P)
Human Human PBMCPBMC
Mouse spleen cell
human SLC with Chemotactic Activity human SLC with Chemotactic Activity for mouse immune cells for mouse immune cells
human SLC with Chemotactic Activity human SLC with Chemotactic Activity for mouse immune cells for mouse immune cells
A and B: Human PBMC; C and D: mouse spleen cell;
A and C: Negative control (200×); B and D: SLC-3P-Fc (200×)
pathology
H&E staining of dermis tissues sections from C57BL/6 mice, non-treated mice (A), treated with pC (B) or pSLC-3P-Fc (C). Images are representative of multiple microscopic fields observed in at least three mice per group.
A B C
A B C
Transfection with pSLC-3P-Fc chemoattracts Transfection with pSLC-3P-Fc chemoattracts lymphocyteslymphocytes to the immunization siteto the immunization site
Transfection with pSLC-3P-Fc chemoattracts Transfection with pSLC-3P-Fc chemoattracts lymphocyteslymphocytes to the immunization siteto the immunization site
Immunotherapy effects of human Immunotherapy effects of human pSLC-pSLC-3P3P-Fc gene vaccine on mouse -Fc gene vaccine on mouse
modelmodel
Immunotherapy effects of human Immunotherapy effects of human pSLC-pSLC-3P3P-Fc gene vaccine on mouse -Fc gene vaccine on mouse
modelmodel
B16-3P tumor cell subcutaneous inoculated in C57BL/6 mice on day0. The tumor bearing mouse immunized by gene gun with different gene vaccine on day 4, day 9 and day 14.
3P: PSA-PAP-PSM; Target: B16-3P;
0
500
10001500
2000
2500
30003500
4000
4500
6 7 8 10 12 14 16 18 20 22 24 26Days after tumor injection
Tum
or v
olum
e(m
m3)
pC
p3P
pSLC
pFc
pSLC- 3P
pSLC- Fc
p3P- Fc
pSLC- 3P- Fc
pSLC+p3P+pFc
0
10
20
30
40
50
60
70
80
90
100
25 30 35 40 45 50 55 60
days af ter tumor i nocul at i on
surv
ival
%
pC
p3P
pSLC
pFc
pSLC- 3Ps
pSLC- Fc
p3P- Fc
pSLC- 3P- Fc
pSLC+p3P+pFc
Induction of human CTL against cancer by CADV Induction of human CTL against cancer by CADV in vitroin vitro
Induction of human CTL against cancer by CADV Induction of human CTL against cancer by CADV in vitroin vitro
Day 0
Day 1
Day 6
Day 13
PBMC HLA-A0201
MLC
CADV transfectedCADV transfected
Restimulate
MLC: Mixed Lymphocyte Culture
0
10
20
30
40
50
60
70
T2 PC-3M MCF7 MCF7-Pc
MCF7-hPSA
MCF7-hPSM
MCF7-hPAP
LNCaP
Cytolysis %
A
B
A0201 + - + + + + + +hPSA - + - - + - - + hPSM - + - - - + - + hPAP - + - - - - + +
SLC-3P-Fc DNA Vaccine induced CTL by SLC-3P-Fc DNA Vaccine induced CTL by MLCMLC
A: Stimulating cell tansfected by pSLC-3P-Fc B: Stimulating cell tansfected by pcDNA
Effector:Target=60:1
Universal Chemo-Antigen DNA VaUniversal Chemo-Antigen DNA Vaccine (ccine (CADV)CADV)
广谱趋化抗原基因疫苗广谱趋化抗原基因疫苗
Enzyme Cut SitesEnzyme Cut SitesEnzyme Cut SitesEnzyme Cut Sites
SLCSLC FcFcCMVCMV
PSA,PAP,PSMPSA,PAP,PSMhTERThTERT
The CTLs induced by pSLC-T-Fc-transfected PBMC, pC-transfected PBMC, and non-transfected PBMC were tested for cytolytic activity against MCF-7, SK-BR3, U2OS/T, U2OS, LNCap, PC-3M, SK-OV-3, and T2 .
SLC-TERT-Fc DNA Vaccine induced human CTL by MLCSLC-TERT-Fc DNA Vaccine induced human CTL by MLCSLC-TERT-Fc DNA Vaccine induced human CTL by MLCSLC-TERT-Fc DNA Vaccine induced human CTL by MLC
0
500
1000
1500
2000
2500
3000
3500
14 17 20 23 26
Days after tumor injection
Tum
or V
olum
es (
mm
3 )
B16/C+anti-4-1BB+Ig
B16/SLC-Te-Fc+anti-4-1BB+anti-CD8
B16/SLC-Te-Fc+anti-4-1BB+anti-CD4
B16/SLC-Te-Fc+anti-4-1BB+anti-asialoGM1
B16/SLC-Te-Fc+anti-4-1BB+Ig
FIGURE 8 – Assessment of involvement of NK cells, CD4+ and CD8+T cells in antitumor effect. Animals were s.c. challenged with 5 x 104 B16/Te cells on day 0, then treated with B16/SLC-Te-Fc or B16/C plus anti-4-1BB (n = 4) as Figure 6a described. Cell subsets were depleted by the injection of GK1.5 (for CD4+ T cells), 2.43 (for CD8+ T cells) ascites fluids (100 ml/mouse) on days 2, 4 and 6, then twice a week before killing. For NK cells depletion, asialo-GM1 antibodies (200g/mouse) were injected for 5 consecutive days beginning on day 2, then twice a week before killing. The control Ig was injected in the same way. (a) Abrogation of CTL-mediated cytotoxicity. (b) The s.c. tumor size was measured twice per week.
Assessment of involvement of NK Assessment of involvement of NK cells, CD4+ and CD8+T cells in cells, CD4+ and CD8+T cells in
antitumor effectantitumor effect
Assessment of involvement of NK Assessment of involvement of NK cells, CD4+ and CD8+T cells in cells, CD4+ and CD8+T cells in
antitumor effectantitumor effect
CD8
CD4
NK
C57BL/6 mice were challenged via tail vein with 1 x 105 B16/Te tumor cells on day 0 (n = 5), and and were treated with CADV modified tumor cells immunization on days 7 and 11 followed by either rat Ig or anti-4-1BB MAbs administration (i.p.) on days 8, 11 and 14. The lung metastatic nodules were counted on day 23. *p < 0.001 versus B16/C + Ig and B16/C + anti-4-1BB by Student paired t-test; **p < 0.05 versus B16/CCL21-Te-Fc 1 Ig by Student paired t-test.
B16/SLC-Te-Fc + anti-4-1BB
B16/SLC-Te-Fc + Ig
B16/C + anti-4-1BB
B16/C +Ig
Evaluation of therapeutic effect on established Evaluation of therapeutic effect on established pulmonary metastases by CADV-tumor + anti-pulmonary metastases by CADV-tumor + anti-
41BB41BB
The Advantageous Features of ChemotactiThe Advantageous Features of Chemotactic-Antigen DNA Vaccinec-Antigen DNA Vaccine
The Advantageous Features of ChemotactiThe Advantageous Features of Chemotactic-Antigen DNA Vaccinec-Antigen DNA Vaccine
1. The CADV could facilitate the co-localization of DC, T, B and NK cells by SLC and induce effective cell-mediated immune response and humoral immune response .
2. CADV fusion protein can be forcefully captured by DC (Fc/FcR). This approach can efficiently increase antigen presentation by DCs, to induce both antigen-specific Th and CTL.
3. Tumor antigen specific memory T cells can be highly induced by CADV to prevent tumor recurrence and metastasis.
4. CADV is a recombinant plasmid DNA. It is suitable for industrialisation.
5. CADV is a vaccine platform. Tumor or non-tumor vaccines can be easily made by this system.
1 、 Specific antitumor immune response induced by a novel DNA vaccine composed of multiple CTL and T helper cell epitopes of prostate cancer associated antigen , Immunology Letters 99:85-93,2005
2 、 Enhancement of Antitumor Immunity by a Novel Chemotactic Antigen DNA Vaccine Encoding Chemokine and ultiepitope of Prostate Tumor Associate Antigens, Immunology , 117: 419-430, 2006
3 、 Enhancement of DNA vaccine potency by sandwiching antigen-coding gene between secondary lymphoid tissue chemokine (SLC) and IgG Fc fragment genes, Cancer Biology and Therapy , Apr 20;5(4), 2006
4 、 Enhanced anti-tumor effect against human telomerase reverse transcriptase (hTERT) by vaccination with chemotactic-hTERT gene-modified tumor cell and its combination with anti-4-1BB monoclonal antibodies, International Journal of Cancer 119, 2006
5 、 Induction of protective and therapeutic antitumour: immunity using a novel tumour-associated antigen-specific DNA vaccine,Immunology and Cell Biology 84, 1–8 , 2006
6 、 Potent Systemic Antitumor Immunity Induced by Vaccination with Chemotactic-Prostate Tumor Associated Antigen Gene-Modified Tumor Cell and Blockade of B7-H1 , Journal of Clinical Immunology, 27(1):117-130, 2007
7 、 Synergistic antitumor effect of chemotactic-prostate tumor associated antigen gene-modified tumor cell vaccine and anti-CTLA-4 mAb in murine tumor model , Immunology Letters, 113:90–98, 2007
8 、 A novel chemotactic-antigen DNA vaccine against cancer, Future Oncology, Apr;4(2):299-303,2008
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Lin ChenSun Wenxin
Qin Hanjun
Liu RongzhiLin Xiaoyan