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