Immunoadjuvant Properties of Oncolytic Schwarz Measles Virus

Dr Jean-François FonteneauInstitut de Recherche en Santé de

l’Université de NantesINSERM UMR892, CNRS UMR6299

Nantes, France

Anti-tumor Virotherapy using oncolytic virus

Oncolytic virus

Ideal replicating oncolytic virus: (Russell SJ, Nature biotechnol, 2012Vacchelli E et al, Oncoimmunology, 2013)

- Kills efficiently tumor cells (apoptosis)Healthy cells

- No toxicity (genetically stable, minor side effects)

- High viral concentration production capability

- Infects exclusively or preferentially tumor cells

Tumor cells

Infection

apoptosis

- Induction or stimulation of an anti-tumor immune response.

Activation of the immune system

Measles virus vaccine (MV) based anti-tumoral virotherapy

Measles virus vaccine, Schwarz strain (MV): Dr Frédéric Tangy (Viral genomics and Vaccination laboratory, Pasteur Institut, Paris)

- 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

- 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)

Protéine L

Protéine de Fusion (F)

Hémagglutinine (H)

Protéine de Matrice (M)

Double membrane lipidique

Phosphoprotéine (P)

ARN + Nucléoprotéine (N)

- Enveloped, non-segmented, negative-sense, single-stranded RNA (ssRNA) paramyxovirus of the genus Morbillivirus

Guillerme JB et al, Biology, 2013

- antiviral pathway defects are often found in tumor cells

Video : MV infection of meso13 by MV-eGFP (enhanced green fluorescent protein)

MV infection of Malignant Pleural Mesothelioma tumor cells

Limited MV infection of healthy mesothelial cells

Video : healthy mesothelial cells exposed to MV-eGFP

Cross presentation to CD8+ T Lymphocytes (CTL)?

Tumor cells

Immature DC

Apoptosis

MV

Activation of the immune system by MV infected tumor cells ?

Danger signalsDAMPs ? PAMPs ?

Mature DC ? Myeloid DC? Plasmacytoid DC?

20

40

60

80

% C

D83

+ cells

5000

10000

IL-12

p70

(pg/

ml)

10

20

Med

ian

Fluo

resc

ence

Inte

nsity

1000

2000

3000

IL-10

(pg/

ml)

CD83

CD86 IL-10

IL-12p70

Mo-DC activation

Activation of monocyte derived DC (Mo-DC) and tumor antigen cross-priming by MV infected tumor cells

Gauvrit et al, Cancer Research, 2008

Tumor antigen cross-priming

• Expression of TLR7 et TLR9 => specialized in recognition of viral nucleic acids

MVARNsb

IFN-Production and tumor antigen cross-presentation by pDC exposed to MV infected tumor cells?

Plasmacytoid Dendritic Cells (pDC)

•Produce huge quantities of Type I IFN (- and –) 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.

pDC are not infected by Schwarz MV

Video : pDC exposed to MV-eGFP

MV infected tumor cells induce maturation of plasmacytoid DC

CD

123

SS

C

CD86 CD40

9 %

92.4 %

20 %

90.6 %

7.42 %

CD83

92.4 %

BDCA4FSC

4,27

130,96

5,13

5,99

12,36

5,33

24

10

60

22

85

167

Meso13 MV

Meso13 UV

IL3

R848

MV

IL-3 + MV

CD83

IL3

MV

MV

+IL

3

R8

48

M18

MV

M18

UV

Mes

o1

3 M

V

Mes

o1

3 U

V

A5

49 M

V

A5

49 U

V

0

20

40

60

80

100

** *****

******

% P

os

itiv

e C

ells

CD40

IL3

MV

MV

+IL

3

R8

48

M18

MV

M18

UV

Mes

o1

3 M

V

Mes

o1

3 U

V

A5

49 M

V

A5

49 U

V

0

50

100

150

200

*

R-M

FI

CD86

IL3

MV

MV

+IL

3

R8

48

M18

MV

M18

UV

Mes

o1

3 M

V

Mes

o1

3 U

V

A5

49 M

V

A5

49 U

V

0

20100

150

200

*

R-M

FI

MV infected tumor cells are internalized by plasmacytoid DC

4°C 37°C

MV

UV

M18

MV

UV

A549

37°C

4°C

PKH

67

BDCA-4

PKH

67

HLA-DR

pDCMo-DC

HLA-DR Alexa568 PKH-67 MERGE

pDC +M18 MV

pDC + A549 MV

MV activates pDC IFN- production by TLR7

IRS661: TLR7inhibitor

IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1

0

20

40

60

80

200

400

600

800

IL-3 + MV*10 CpG-A CpG-AIF

N- (

ng

/ml)

IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1

0

20

40

60

80

200

400

600

800

IL-3 + MV*10 CpG-A CpG-A

IF

N-

(n

g/m

l)

IRS661

IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1

0

20

40

60

80

200

400

600

800

IL-3 + MV*10 CpG-A CpG-A

IFN

- (

ng

/ml)

IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1

0

20

40

60

80

200

400

600

800

IL-3 + MV*10 CpG-A M18 MV

IFN

- (

ng

/ml)

IRS661 IRS661

(TLR-9)

pDC

IFN

-α (n

g/m

l)

IL3

MV

IL3

+ M

V

R8

48

M1

8 M

V

M1

8 U

V

A5

49

MV

A5

49

UV

0

10

20

30

200

400

600

IFN

- (

ng

/ml)

MV probably activates pDC via TLR7 in early endosome

CpG-A (TLR9)

Kerkmann M et al, J Immunol, 2003

CpG-B (TLR9)

-> High IFN- -> Low IFN-

-> Low inflammatory molecules secretion (IL-8)

-> High inflammatory molecules secretion (IL-8)

-> Low costimulation (CD80, CD86)

-> High costimulation (CD80, CD86)

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-B 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

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

NYESO-1

M 18 A5490

5000

10000

15000NIUVMV

(NY

ES

O-1

/ R

PL

PO

)*10

00

M18 : HLA-A*0201- , NYESO-1+

A549 : HLA-A*0201- , NYESO-1-

pDC : HLA-A*0201+ , NYESO-1-

M117.167 : HLA-A*0201/NYESO-1(157-165) specific CD8+ T cell clone

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

LT only pDC + LT

0.1µM NYESO-1[157-165]

1µM NYESO-1[157-165]

M18

MV UV

M18 MV

A549

R848

M18 UV

10.8%

6.5%

0.8% 0.2%

0.2%

87.5%

0.1% 0.1%

0.3% 0.1%

LT - pDC

CD8

IFN-γ

CD8

IFN-γ

LT + pDCCD

8

IFN-γ

CD8

IFN-γ

CD8

IFN-γ

CD8

IFN-γ

LT

on

ly (

n=

5)

pD

C +

LT

(n

=6

)

pD

C 0

.1 µ

M N

YE

SO

-1 (

n=

3)

pD

C +

M1

8 M

V (

n=

6)

pD

C +

M1

8 U

V(n

=5

)0

10

20

% o

f IF

N +

T C

D8+

cel

ls

NYESO-1 cross-presentation by pDC and Mo-DC exposed to MV infected tumor cells

HLA-A*0201neg/NY-ESO-1pos

HLA-A*0201neg/NY-ESO-1pos

Mo-DC pDC

Ø

NYESO-1 [157-165](10µM)

M18MV

M18 UV

IFN-y IFN-y

0.6%

11.7%

68%

0.16%

0.3%

8.15%

79.6%

0.4%

CD8CD8

LT

on

ly

Mo

-DC

i

Mo

-DC

m +

0

,1µ

M N

Y-E

SO

-1

Mo

-DC

+ M

18M

V

Mo

-DC

+ M

18U

V0

5

10

15

20

% o

f IF

N +

T C

D8

+ c

ells

Mo- DC Cross- presentation

LT

on

ly (

n=

5)

pD

C +

LT

(n

=6

)

pD

C 0

.1 µ

M N

YE

SO

-1 (

n=

3)

pD

C +

M1

8 M

V (

n=

6)

pD

C +

M1

8 U

V(n

=5

)0

10

20

% o

f IF

N +

T C

D8+

cel

ls

pDC cross-presentation

0,5

Mo-DC

95,1

Mo-DC + pep 1µM

M18 MV

M18 UV

Mo-DC Mo-DC + IFN-γ + TNF-α

Mo-DC + R848

10,0 22,7 7,0

0,5 2,2 0,9

M18 MV

M18 UV

1,2 4,9 1,3

0,5 2,3 0,7

(-) IFN-γ + TNF-α R848

0

10

20

30

% C

D8+ /

IFN

γ+ cel

ls

Mo-

DC (-)

R848

IFN

-γ T

NF-

(-)

R848 (-)

R848 (-

)

R848

M18 MV M18 UV M18 MV M18 UV

(+) Mo-DC (-) Mo-DC

IFN

-γ T

NF-

IFN

-γ T

NF-

IFN

-γ T

NF-

Deauvieau F et al, Int J Cancer, 2014

NYESO-1 cross-presentation by Mo-DC is increased by TNF-/IFN-

Conclusions

PreferedInfection

Limited infection

MV

Tumor cells(CD46high)

Healthy cells(CD46low)

Apoptosis

Immature Mo-DC

Immature plasmacytoid DC

MV-infectedTumor cells

Mature Mo-DC

Mature plasmacytoid DC

TAAPAMP?DAMP(HSP70, gp96)

TAAPAMP(MV Single-strand RNA)DAMP?

Phagocytosis

Phagocytosis

Maturation

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-

Gauvrit A et al, Cancer Research, 2008

Guillerme JB et al, Clinical Cancer Research, 2013

Donnelly OG et al, Gene Ther, 2013

(IFN-, IFN-,HMGB1, IL-6IL-8)

Blood myeloid DC ? CD1c+ DC ? CD141+ DC ?

Plateforme de dévelopement et de transfert à la clinique (CHU Nantes) :Delphine Coulais, ingénieurClarisse Panterne, ingénieur

Service de pneumologie du CHU de NantesLaurent Cellerin, Christine SaganAntoine Magnan

Service de pneumologie du CHU de LilleArnaud Scherpereel

Institut Pasteur, Viral genomics and Vaccination laboratory Frédéric TangyMariana Mesel-Lemoine

Collaborations

Centre de Recherche en cancérologie de LyonJenny ValladeauChristophe Caux

Marc Grégoire, INSERM Christophe Blanquart, CNRSDaniel Pouliquen, INSERMJean-François Fonteneau, INSERMNicolas BoisgeraultJean-Baptiste GuillermeCarole Achard, Ferdaous AllaguiIza DenisClarisse PanterneSophie Deshayes

Team (Inserm U892,Nantes)

Funding