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Thérapie cellulaire et génique pour letraitement des cellules tumorales
Pr Francois Lemoine - CHU Pitié Salpêtrière - CNRS UMR 7087
Réponse Immune Antitumorale
NK
CTL
T4
T8
Celluletumorale
B
MØ
APC
Réponse ImmunitaireInnée
Réponse ImmunitaireAdaptative
DC
TEff TReg
HSC
Interactions DC - LT effecteurs et LT régulateurs
Quelles stratégies ?
•Cellules dendritiques•Lymphocytes T régulateurs•Destruction conditionnelle des cellules tumorales
Dendritic cellsAnti-tumoral immunotherapy
Dendritic cells (DC)
1868 : Description in the skin by Paul Langerhans
1973 : Description in murine lymphoid organs by Ralph Steinman and Zanvil Cohn
defined as “professional” antigen presenting cells (APC)
Blood
Draining lymph nodes
Lymph
Liver
Intersticial spaces(Kidney, lung, heart..)
CDprecursors
Epithelia (skin, mucosae..)
Bone marrow
DC-LC
Veiledcells l
IDC
Interstitial DC
Plasmacytoid cellsGCDC
DC CD11c+DC CD11c- or plasmacytoid
DC CD83+
Veiled cells
Efferent lymph
Afferentlymph
Medulla
Primary folliculeParacortexHEV
Cortex
Distribution of dendritic cells
Functions of dendritic cells
Lymph node T cell area Tcells
LymphEpiderm
Langerhanscells
Ag.
Blood
Antigen uptake
Presentationto T cells
Bonemarrow
CD 34 +
Migration
Veiledcells
interdigitatingcells
Morphology and immunophenotypic markersof dendritic cells
Homing moleculesCD11a, b, c
CD49d, CD44 variantsE-cadherin
Adhesionand costimulatory molecules
CD50, CD54/ICAM-1,3CD58/LFA3CD80, CD86
Virus receptorsCD4, CD46
Chemokine-RDC-SIGN
Other markersCD83
DC-Lamp, Langerin
Cytokine and chemokine receptorsTNF-R : CD120, CD40
Cytokine-R : GM-CSF, IL1, IL10, IL4, TGFβ
Chemokine-R :CCR5, CCR6, CXCR4
Molecules for antigen presentation
MHC I, MHC II, CD1Receptors for
Antigen uptakeMMR, DEC205
FcγR(CD32, CD64), CD36αvβ5, CR
W. Michael McDonnell et al (1996)The new England Journal of medecine, vol.334, n°1
W. Michael McDonnell et al (1996)The new England Journal of medecine, vol.334, n°1
DC
T4
B
CD80 CD86
CD54
CD58
CD11a
CD8
CD80 CD28
CD40CD40 L
CD28
CD80
CD40
CD40 L
CD19
CD20
CD40 L
Ig GIg A
1
3
21
TCR
MHC II
MHC I
1
2
3
T8
NK
IL-12
DC and Immunotherapy : the concept
Reinjecting ex vivo-pulsed DC represents a potentially powerful
tool to elicit immunity against viral or tumour-associated Ag.
DC-based anti-tumoral vaccination in mice
Exogeneous antigenstumor extracts Nair, 1997
native antigens Celluzi, 1996 &Pargador, 1996
acidic eluatsZitvogel, 1996
apoptotic bodiesAlbert,1998
exosomesZitvogel, 1998
Gene transfer
adenovirusSong, 1997
retrovirusSpecht, 1997
RNABoczkowski, 1996
immaturedendritic cell
naked DNACondon, 1996
tumor regression
DC progenitors orDC precursors
DC loadedwith Ag of
interest
Sensitization
GrowthFactors
Injection
Use of DCs in Immunotherapy
Ι
ΙΙ
ΙΙΙ
ΙV
MOELLE
OSSEUSE
SANG
T ISSU
? ?
Thymus
Cellule souche
Cellule souche myéloïde Cellule souche lymphoïde
CFU-GEMM
BFU-E
CFU-E
BFU-MK
CFU-MK
CFU-GM
CFU-G CFU-M CFU-DC
CFU-Eo CFU-BaE
Myélob Monob
Poly N MonoPoly BaPoly EoPlaqGR
Mast DC Plas T4 T8
T4 T8
T4 T8
B
B
NK
MΦ
Meg
Pro-B
Poly Eo
Production of dendritic cells from monocytes or CD34+ cells
MonocyteGM-CSF / IL4
d7
CD34+Flt3-L / SCF
GM-CSF / TNF α d5
+ IL4LC-DC
d12
d8
+ IL4
Mφ-DC
Immature DC
Low expression of MHC andcostimulatory molecules
Weak immunostimulation of T lymphocytes
High ability for antigen uptake and processing Weak ability for antigen uptake and processing
High expression of MHC andcostimulatory molecules
High immunostimulation of T lymphocytes
LPS, TNFα, CD40-L
Mature DC
Properties of mature dendritic cells
Authors
Hsu,1996
Murphy,1999
Kugler,2000
Höltl2002
Pathology
Follicular lymphoma
(stage IIIA & IVA)
Prostate carcinoma
RenalCarcinoma
RenalCarcinoma
n
4
37
17
27
Antigen
Idiotypic proteins
HLA- restricted peptides
PSM-P1 or P2
Allogeneic DC/ tumor cell hybrids
Autologous tumor lysatsor cell line
Injection route
IV
IV
Intradermal
IV andIntradermal
4 / 4Ag-specific
proliferation
4 / 37
11 /17DTH
14/27DTH/KLH
KLH-proliferation
1 CR1 PR
1 CR10 PR
4 CR, 2 PR1 MR
2 CR1 PR7 SD
Origin of DCs
Monocytes
Monocytes
Monocytes
Monocytes
ResponseBiologicalBiological ClinicalClinical
DC-based Immunotherapy : Clinical Trials (I)
Authors
Chakraboy1998
Nestlé,1998
Thurner,1999
Mackensen,2000
Lau,2001
Banchereau2001
Pathology
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
n
13
16
11
14
16
18
Antigen
Tumor lysates
Pool of peptidesor tumor lysates
HLA- A1 restricted Mage-3 peptides
HLA- restricted peptides
HLA- restricted peptides : GP100
TyrosinaseMART-1/Melan A
GP100Tyrosinase
MART-1, Mage 3 HLA- restricted
peptides
Injection route
Intradermal
Intra-lymph nodes
Subcutaneous +Intradermal + IV
IV
IV
Subcutaneous
9 / 13DTH
11 / 16DTH
7 / 11 DTH8 / 11 CTL
4 / 14 DTH1 / 14 CTL
12 / 16 T-cell proliferation
5 /16 Elispot
15/18 DTH/KLH10/14 DTH/peptide8/18 Ag-specific
Elispot
1 PR
2 CR, 3 PR1 MR
3 CR3 PR
1 CR1 PR
1 CR4 PR
3 CR4 PR3 SD
Origin of DCs
Monocytes
Monocytes
Monocytes
CD34+ cells
Monocytes
CD34+ cells
ResponseBiologicalBiological ClinicalClinical
DC-based Immunotherapy : Clinical Trials (II)
tumor lysates
400ml Bone Marrow
CD34+
d0
CD34++ KLH - Clinical tolerance
- Biological tolerance- Clinical response- Immunological responseLysates
Follow up :
Dendritic cells
d15 - 45
d22 - 53
d30 - 60
d60 - 90
d90 - 120
IL2 sc W1
W2
W4
W5
W7
W8
W10
W11
d60 d90 d120 d150DC-based Immunotherapy of Renal Carcinoma
Preliminary resultsPatients UPN CD34+
Production
Lysates
Production
Vaccination
Number
Biological
responses
Clinical outcome
p201 Yes Yes 5 IFNg
&
Perforine
production
Dead 18 month
after
treatment
p101 No Yes No NA Dead;
excluded from
protocol
p401 Yes Yes 3 NA Dead: clinical
progression
p502 Yes Yes No NA Dead: clinical
progression ;
excluded
before
treatment
p501 Yes Yes 3 NA Dead: Clinical
Progression
p402 Yes Yes 5 IFNg
&
Perforine
production
Alive 16 month
after
treatment
tumor extractsPeptidesExosomesApoptotic bodies
Sensitization
Purification Differentiation
CD34+/Monocytes
Endogeneous sensitization pathway exogeneous sensitization pathway
DCGene transfer
Viral / non-viral vectors
TransducedDC
DCdifferentiation
CD34+ /Monocytes
DC
Dendritic cells-based immunization in human
tumor cellsInjection
Sensitized DC
TransducedCD34+ /
Monocytes
Dendritic Cell
Tumor Associated Ag(TAA) Loaded dendritic cell
Strategies for DC-based immunotherapy of cancer
CTL
CTL
Gene codingFor TAA Transduced DC
Viral particles
+
=+
Gene coding For TAA
In vivo injection
In vivo transduction of DC
+
GENETIC IMMUNOTHERAPY USINGDENDRITIC CELLS
Source of DCMonocytesCD34+ cells
Injection of DC or injection of recombinantlentiviral particles
Evaluation of the anti-tumor immuneresponse
CELLULES DENDRITIQUES ET IMMUNOTHERAPIEGENIQUE
Perspectives
• Mélanomes malins métastatiques• Leucémie myéloïde chronique• Evaluation in vitro de la réponse immune• Evaluation in vivo de la réponse immune
dans un modèle murin de souris HLA-A2transgénique
LENTIVIRUS RECOMBINANT
VECTEUR LENTIVIRAL- Séquences codantes pour le gène d ’intérêt- Signaux en cis nécessaire à la RT, intégration et transcription et à l ’encapsidation du vecteur
Gène d’intérêt : gène thérapeutique et/ou gène rapporteur
LTR GA RRE P Thy-1 IRES eGFP WPRE
LTRsin
EF1∝-L
EF1∝-S
CMV
ψ
Vecteurs lentiviraux bicistroniques et DCs
NI
EF1LW+
CMVW+
EF1LVpx+
CMVVpx+
CMVVpx-
EF1SW+
EF1S
CMV
99%
99%
98%
97%
98%
85%
85%
77%
41%
28%
2% 2%
96%
99%
99%
98%
94%
77%
CD90 GFPCD90 GFP
59%
59%
56%
58%
60%
2%
14%
16%
14%
70%
64%
56%
59%
62%
2%
48%
81%
40%
HIV vectors
SIV vectors
A : CD34-DC B : Mo-DC
RSV R U5 GA RREΨ
pCMV Thy-1 IRES CML.A2PEs1-4 WPRE
LTR SINcPPT
Exemple de construction poly-épitopique : pHIV-CMV-Thy1-IRES-CML.A2PEs1-4
Vaccination poly-épitopique avec vecteurs lentiviraux
Mélanome : Mart1, Mage 3, gp100, tyrosinaseLMC : Bcr-Abl, WT1, PR1, Tert
DC-based genetic immunotherapy of cancerIn vivo targetting of DCs
CTL
CTL
Viral particles
=
+
Gene coding For TAA
In vivo injection
In vivo transduction of DCTumor cells
Use of lentiviral particles pseudotyped with targetting enveloppe
DC-based genetic immunotherapy of cancerIn vivo targetting of DCs
CTL
CTL
Viral particles
=
+
Gene coding For TAA
In vivo injection
In vivo transduction of DCTumor cells
Use of lentiviral particles pseudotyped with targetting enveloppe
Depletion ofregulatory/suppressor T-cells
for inducing anti-tumor immune response
Role of regulatory/suppressor T-cells
•Maintenance of self tolerance & prevention of auto-immune disease•Defect of Treg cells in HCV with vasculitis-induced cryoglobulinemia
•Tolerance induction to allogeneic transplants•Treg depletion allows control of GVHD
•Maintenance and/or physiopathology of chronic infections•Role in anti-tumor immunity
•Increase number of Treg in lung , breast, gastrointestinal tumors•Treg depletion improves anti-tumor immune response
Depletion of regulatory/suppressorT-cells
•Injection of anti-CD25 monoclonal antibodies ± Cyclophosphamide•Validation in murine tumor models
Tumor rejection by in vivo administration of anti-CD25mAb
Days
Mea
n tu
mor
dia
met
er (m
m)
Onizuka et al. (1999) Cancer Research 59:3128
Specific targetting andconditional destruction of tumor cells
Objectives
•Use of lentiviral particles pseudotyped with targetting enveloppe•Transduction of tumor cells with suicide gene•Stimulation of immune cells
Pseudotyped lentiviral particlesExample : B-cell lymphoma
CD20 mAbs
Tumor B-cells
Suicide geneThe thymidine kinase / ganciclovir system
GCVHSV-TK
GCV-MPCellular
kinaseGCV-TP
Incorporationinto
elongatingDNA
Death ofdividing cells
N
N
OHN
NH2N
OHOOH
Transduced cells
GCV
Dividing cells
Suicide gene and immunostimulation
RSV R U5 Immuno-stimulationGA RREΨ
pCMV HSV1-TK IRES WPRE
LTR SINcPPT
CD40/CD80
GM-CSF
IL-12
Pseudotyped particles
In vivo transduction of lymphoma cells
5’ LTR ψ IRES 3’ LTR
Lentiviral construct
Patient
HSV1-TK IS Gene
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