Measles virus vaccine induces oncolysis of

tumor cells and activates immune responses.

Marc Grégoire*, J.-F. Fonteneau, N. Boisgerault, J.-B. Guillerme and F. Tangy¤.

* INSERM, Nantes, F-44000, France.

¤ Pasteur Institute, Viral Genomics and Vaccination, Paris, France

Oncolytic

virus

Ideal replicating oncolytic virus:

Russell SJ, Nature biotechnol, 2012

Vacchelli E et al, Oncoimmunology, 2013

- Kills efficiently tumor cells (apoptosis)

Infection

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

Anti-tumor Virotherapy using oncolytic virus

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

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

Gauvrit A et al, Cancer Research, 2008; Boisgerault N et al, Biomed Res Int, 2013

Oncovirotherapy – Oncolytic properties of Measles Vaccine

Virus MV Oncolytic (Vaccine strain)

Oncolytic properties

Immunogenic apoptotic cell

death

Tumour Cell

T Lymphocytes Plasmacytoïd DC Myéloïd DC

Specific T cell

response

immunogenic molecules

DAMP & PAMP

Tumour cell infection (mesothelioma)

INFECTION

Syncitium

MV-eGFP Infected cells Mesothelioma

0 24 48 720

20

40

60

80

100

Meso13Meso47Meso56Meso96

Meso11

Time post infection (h)

% e

GFP

+ce

llsOthers

•Mesothelioma : 14/18

• Melanoma : 7/8

• Lung : 3/4

• Colorectal : 4/4

Mesothelioma (Gauvrit A et al, Cancer Research, 2008)

Lung, Colorectal adenocarcinoma (N. Boisgerault et al.,

Biomed. Res. Int., 2013)

Mesothelial cells

Normal cells: mesothelial and lung cells

Lung cells

Also for :

• Fibroblasts

• Endothelial cells

Oncovirotherapy - Live attenuated Schwarz Measles Vaccine

T Lymphocytes

Virus MV Oncolytic (Vaccine strain)

Plasmacytoïd DC

Oncolytic properties

Immunogenic apoptotic cell

death

Myéloïd DC

Specific T cell

response

Tumour Cell

immunogenic molecules

DAMP & PAMP

Danger Signals: Damage Associated Molecular Patterns

Meso13 A549 M6 HT29

100

200 ControlMV

Me

dia

n F

luo

resc

en

ce In

ten

sity

HSP70

Cross-presentation of

tumor-derived antigens

Meso13 ADK153 M6 HT29

25

50

% C

alr

eti

culi

n+ c

ell

s

Calreticulin

Phagocytosis

HMGB1 Antigen processing and

presentation

Meso13 A549 M6 HT29

100

200

300

400

HM

GB

1 (

ng/

mL)

Bianchi and Manfredi, 2007 Kepp et al., 2009

Gregoire et al, in preparation

Immune response with Live attenuated Schwarz Measles Vaccine

T Lymphocytes

Virus MV Oncolytic (Vaccine strain)

Plasmacytoïd DC

Oncolytic properties

Immunogenic apoptotic cell

death

Myéloïd DC

Specific T cell

response

Tumour Cell

immunogenic molecules

DAMP & PAMP

Activation

maturation

Plasmacytoid dendritic cells Myeloid dendritic cells

Mo-DC

pDC

MV preserves dendritic cells

(Guillerme JB et al., 2013, Clinical Cancer Research) (Gauvrit A et al., 2008, Cancer Research)

MV-infected tumor cells induced Mo-DC Maturation

HLA-A,B,C

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

1000

2000

MF

I

HLA-DR

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

50

100

MF

I

CD80

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

100

200

MF

I

CD83

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

50

100

% C

D8

3+ ce

lls

CD86

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

200

400

600

MF

I

CD40

Dci

IFN

TNF

M11

UV

M11

MV

M13

UV

M13

MV

0

200

400

600

800

1000

MF

I

DCi

IFNα

- P

oly

I:C

TN

Fα

- P

oly

I:C

Meso 11 Meso 13

MV UV MV UV

CMH I

CD86 CMH II

CD83 CD80

CD40

DCi

IFNα

- P

oly

I:C

TN

Fα

- P

oly

I:C

Meso 11 Meso 13

MV UV MV UV DCi

IFNα

- P

oly

I:C

TN

Fα

- P

oly

I:C

Meso 11 Meso 13

MV UV MV UV

(Gauvrit A et al., 2008, Cancer Research)

MUC-1 or mesothelin +Mo-DC

CD8

DCi IFN -PolyI:C UV MV0

1000

2000

3000

4000

cp

m

CD4

DCi IFN -PolyI:C UV MV0

50000

100000

cp

m

Meso 13 Meso 13

0

50

100

% C

D8+

IF

N-

+

TNFα-

PolyI:C

Meso 13 MV DCi

T CD4 Proliferation T CD8 Proliferation

Clone T CD8 anti-MUC-1 Cross-presentation (mesothelin)

(Gauvrit et al., 2008) (Gauvrit A et al., 2008, Cancer Research)

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

MV

ARNsb

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

infected tumor cells?

•Produce huge quantities of Type I IFN (- 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.

Plasmacytoid dendritic cells

4°C 37°C

MV

UV

M18

MV

UV

A549

37°

C

4°C

PK

H67

BDCA-4

PK

H67

HLA-DR

pDC Mo-DC

HLA-DR Alexa568 PKH-67 MERGE

pDC +

M18 MV

pDC +

A549 MV

MV infected tumor cells are internalized by Plasmacytoid dendritic cells

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

R84

8

M1

8 M

V

M1

8 U

V

Me

so

13

MV

Me

so

13

UV

A5

49

MV

A5

49

UV

0

20

40

60

80

100

** *** **

*** ***

% P

os

itiv

e C

ell

s

CD40

IL3

MV

MV

+IL

3

R84

8

M1

8 M

V

M1

8 U

V

Me

so

13

MV

Me

so

13

UV

A5

49

MV

A5

49

UV

0

50

100

150

200

*

R-M

FI

CD86

IL3

MV

MV

+IL

3

R84

8

M1

8 M

V

M1

8 U

V

Me

so

13

MV

Me

so

13

UV

A5

49

MV

A5

49

UV

0

20

100

150

200

*

R-M

FI

MV infected tumor cells induce Plasmacytoid dendritic cells maturation

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-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 CpG-A

IFN

- (

ng

/ml)

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)

pD

C IF

N-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 infected tumor cells induce production of INF type I by pDC

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

CD

8

IFN-γ

CD

8

IFN-γ

LT + pDCC

D8

IFN-γ

CD

8

IFN-γ

CD

8

IFN-γ

CD

8

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+

cell

s

MV infected tumor cells induce NY-ESO-1 cross-presentation by pDC

Cytotoxic

CD8+ T Lymphocytes (CTL)

Tumor cells

Dendritic cells

Apoptosis

MV

Gauvrit A et al, Cancer Research, 2008

Immune system activation

• Activation of myeloïd DC

• Activation of tumor antigen specific

CD8+ T lymphocytes

Dangers signal expression

• HMGB1

• Calreticuline

• HSP

• inflammasome, caspase1, IL-1b

Boisgerault N et al, in preparation

Immune system activation

• Activation of plasmacytoid DC

(production of type I interferon)

Guillerme JB et al, Clin. Cancer Res., 2013

In conclusion: Virotherapy and DC activations

IMMUNOGENIC PROPERTIES OF DEAD CELLS

CANCER RESEARCH CENTER, NANTES, FRANCE

MARC GREGOIRE

JEAN-FRANÇOIS FONTENEAU

ANNE GAUVRIT

JEAN-BAPTISTE GUILLERME

NICOLAS BOISGERAULT

VIRAL GENOMICS AND VACCINATION LABORATORY

PASTEUR INSTITUTE, PARIS, FRANCE

FRÉDÉRIC TANGY

MARIANA MESEL-LEMOINE

“DEVELOPMENT AND CLINICAL TRANSFER”

CORE FACILITY PLATFORM

DELPHINE COULAIS

CLARISSE PANTERNE