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