1. Immunoadjuvant Properties of Oncolytic Schwarz Measles Virus
Dr Jean-François Fonteneau
Institut de Recherche en Santé de l’Université de Nantes
INSERM UMR892, CNRS UMR6299
Nantes, France

2. Activation of the immune system
Anti-tumor Virotherapy using oncolytic virus
- Infects exclusively or preferentially tumor cells
Tumor cells
Infection
apoptosis
- Induction or stimulation of an anti-tumor immune response.
Activation of the immune system
Oncolytic
virus
Ideal replicating oncolytic virus:
(Russell SJ, Nature biotechnol, 2012
Vacchelli E et al, Oncoimmunology, 2013)
Infection
Healthy cells
- No toxicity (genetically stable, minor side effects)
- Kills efficiently tumor cells (apoptosis)
- High viral concentration production capability

3. Measles virus vaccine (MV) based anti-tumoral virotherapy
Protéine L
Protéine de Fusion (F)
Hémagglutinine (H)
Protéine de Matrice (M)
Double membrane lipidique
Phosphoprotéine (P)
ARN + Nucléoprotéine (N)
Guillerme JB et al, Biology, 2013
Measles virus vaccine, Schwarz strain (MV):
Dr Frédéric Tangy (Viral genomics and Vaccination laboratory, Pasteur Institut, Paris)
- Enveloped, non-segmented, negative-sense, single-stranded RNA (ssRNA) paramyxovirus of the genus Morbillivirus
- Attenuated replicating vaccine strain of measles virus: Schwarz (MMR vaccine)
- Targets CD46, complement regulatory protein (wt MV targets CD150/SLAM)
- CD46 is expressed at low level by healthy cells
- CD46 is often found overexpressed on tumor cells
- antiviral pathway defects are often found in tumor cells
- Spontaneously oncolytic:
- Lymphoma, glioma, breast, ovary, prostate … (Russell SJ, Mayo Clinic, USA) - MPM, colon and lung adenocarcinoma
(Gauvrit A et al, Cancer Research, 2008; Boisgerault N et al, Biomed Res Int, 2013)

4. MV infection of Malignant Pleural Mesothelioma tumor cells
Video : MV infection of meso13 by MV-eGFP (enhanced green fluorescent protein)

5. Limited MV infection of healthy mesothelial cells
Video : healthy mesothelial cells exposed to MV-eGFP

6. CD8+ T Lymphocytes (CTL)?
Activation of the immune system by MV infected tumor cells ? MV
Cross presentation to
CD8+ T Lymphocytes (CTL)?
Tumor cells
Immature DC
Apoptosis
Danger signals
DAMPs ?
PAMPs ?
Mature DC ?
Myeloid DC?
Plasmacytoid DC?

7. Gauvrit et al, Cancer Research, 2008
Activation of monocyte derived DC (Mo-DC) and tumor antigen cross-priming by MV infected tumor cells
CD83
CD86
IL-10
IL-12p70
Mo-DC activation
Gauvrit et al, Cancer Research, 2008
Tumor antigen cross-priming

8. Plasmacytoid Dendritic Cells (pDC)
Expression of TLR7 et TLR9
=> specialized in recognition of viral nucleic acids MV
ARNsb
Produce huge quantities of Type I IFN (-a and –b) in response to virus
Antigen cross presentation in human:
HIV (Hoeffel, G,.Immunity, 2007) (Crozat, K., J Exp Med, 2010)
Influenza (Lui, G., PLoS One, 2009)
Antigen cross presentation in mice:
Cross-tolérance (Goubier, A., Dubois, B., Immunity, 2008)
OVA (Mouries, J., Blood, 2008)
One of the target of imiquimod (R837, TLR7 ligand) in the treatment of basal cell carcinoma with Aldara.
IFN-a Production and tumor antigen cross-presentation by pDC exposed to MV infected tumor cells?

9. pDC are not infected by Schwarz MV Video : pDC exposed to MV-eGFP

10. MV infected tumor cells induce maturation of plasmacytoid DC
CD83
IL3 MV
MV+IL3
R848
M18 MV
M18 UV
Meso13 MV
Meso13 UV
A549 MV
A549 UV 20 40 60 80
100 **
***
% Positive Cells
CD40 50
150
200 *
R-MFI
CD86
IL3
R848 MV
IL-3 + MV
Meso13 MV
Meso13 UV

11. MV infected tumor cells are internalized by plasmacytoid DCUV
M18
A549
PKH67
BDCA-4
HLA-DR
pDC
Mo-DC
HLA-DR Alexa568
PKH-67
MERGE
pDC +
M18 MV
A549 MV

12. MV activates pDC IFN-a production by TLR7
IRS661: TLR7inhibitor
(TLR-9)
pDC IFN-α (ng/ml)

13. MV probably activates pDC via TLR7 in early endosome
Kerkmann M et al, J Immunol, 2003
CpG-B (TLR9)
-> High IFN-a
-> Low IFN-a
-> Low inflammatory
molecules secretion (IL-8)
-> High inflammatory
-> Low costimulation
(CD80, CD86)
-> High costimulation
CpG-A (TLR9)
Early endosome
Late endosome
Honda K et al, Nature, 2005
Guiducci C et al, J Exp Med, 2006
Sadaka C et al, Blood, 2009
Kubo-Murai M et al, Blood, 2008
IRF7 pathway
NF-kB pathway
HIV (TLR7)
R848 (TLR7/TLR8)
CpG-B (TLR9)
O’brien M et al, J Clin Invest, 2011
MV (TLR7)
R848 (TLR7/TLR8)
Guillerme JB et al, clin cancer Res, 2013

14. NYESO-1 cross-presentation by pDC exposed to MV infected tumor cells
M18 : HLA-A*0201- , NYESO-1+
A549 : HLA-A*0201- , NYESO-1-
pDC : HLA-A*0201+ , NYESO-1-
M : HLA-A*0201/NYESO-1( ) specific CD8+ T cell clone

15. NYESO-1 cross-presentation by pDC exposed to MV infected tumor cells

16. NYESO-1 cross-presentation by pDC and Mo-DC exposed to MV infected tumor cells
LT only
Mo-DCi
0,1µM NY-ESO-1
Mo-DCm +
Mo-DC + M18MV
Mo-DC + M18UV 5 10 15 20
% of IFN g
+ T CD8+ cells Mo -
DC Cross
presentation
pDC cross-presentation
0.6%
11.7%
68%
0.16%
0.3%
8.15%
79.6%
0.4% Ø
NYESO -
1 [157 -
165]
(10µM)
M18 MV
HLA-A*0201neg/NY-ESO-1pos
M18 UV
HLA-A*0201neg/NY-ESO-1pos
CD8
CD8
IFN - y
IFN - y

17. NYESO-1 cross-presentation by Mo-DC is increased by TNF-a/IFN-g
0,5
Mo-DC
95,1
+ pep 1µM
M18 MV
M18 UV
Mo-DC +
IFN-γ + TNF-α
R848
10,0
22,7
7,0
2,2
0,9
1,2
4,9
1,3
2,3
0,7
(-)
% CD8+/IFNγ+ cells
Mo-DC
(-)
R848
IFN-γ TNF-a
M18 MV
M18 UV
(+) Mo-DC
(-) Mo-DC
Deauvieau F et al, Int J Cancer, 2014

18. ->Cross-presentation of TAA to specific CD8+ T cells
Conclusions
Blood myeloid DC ? CD1c+ DC ?
CD141+ DC ?
Gauvrit A et al, Cancer Research, 2008
Prefered
Infection
Limited
infection MV
Tumor cells
(CD46high)
Healthy cells
(CD46low)
Apoptosis
Immature
Mo-DC
plasmacytoid DC
MV-infected
Mature
TAA
PAMP?
DAMP
(HSP70, gp96)
PAMP
(MV Single-strand RNA)
DAMP?
Phagocytosis
Maturation
->Cross-priming of
TAA-specific CD8+
T cell response
->Cross-presentation of
TAA to specific CD8+ T cells
Cross-priming ?
->Production of a large
quantity of IFN-a
Donnelly OG et al, Gene Ther, 2013
(IFN-a, IFN-b,
HMGB1, IL-6
IL-8)
Guillerme JB et al, Clinical Cancer Research, 2013

19. Funding Collaborations Team (Inserm U892,Nantes)
Plateforme de dévelopement
et de transfert à la clinique (CHU Nantes) :
Delphine Coulais, ingénieur
Clarisse Panterne, ingénieur
Service de pneumologie
du CHU de Nantes
Laurent Cellerin,
Christine Sagan
Antoine Magnan
du CHU de Lille
Arnaud Scherpereel
Institut Pasteur, Viral genomics and Vaccination laboratory
Frédéric Tangy
Mariana Mesel-Lemoine
Collaborations
Centre de Recherche en cancérologie de Lyon
Jenny Valladeau
Christophe Caux
Team (Inserm U892,Nantes)
Marc Grégoire, INSERM
Christophe Blanquart, CNRS
Daniel Pouliquen, INSERM
Jean-François Fonteneau, INSERM
Nicolas Boisgerault
Jean-Baptiste Guillerme
Carole Achard,
Ferdaous Allagui
Iza Denis
Clarisse Panterne
Sophie Deshayes