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Immunoadjuvant Properties of Oncolytic Schwarz Measles Virus

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. Anti-tumor Virotherapy using oncolytic virus. apoptosis. Oncolytic virus.

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Immunoadjuvant Properties of Oncolytic Schwarz Measles Virus

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

  3. Measles virus vaccine (MV) based anti-tumoral virotherapy Hémagglutinine (H) • Guillerme JB et al, Biology, 2013 Protéine de Fusion (F) Protéine de Matrice (M) Protéine L Phosphoprotéine (P) Measles virus vaccine, Schwarz strain (MV): Dr Frédéric Tangy (Viral genomics and Vaccination laboratory, Pasteur Institut, Paris) ARN + Nucléoprotéine (N) Double membrane lipidique • - 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 • - Spontaneouslyoncolytic: • -Lymphoma, glioma, breast, ovary, prostate … (Russell SJ, Mayo Clinic, USA)- MPM, colon and lungadenocarcinoma • (Gauvrit A et al, Cancer Research, 2008; Boisgerault N et al, BiomedRes 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. Activation of the immune system by MV infected tumor cells ? MV Tumor cells Apoptosis • Danger signals • DAMPs ? • PAMPs ? Cross presentation to CD8+ T Lymphocytes (CTL)? Immature DC Mature DC ? Myeloid DC? Plasmacytoid DC?

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

  8. Plasmacytoid Dendritic Cells (pDC) • Expression of TLR7 et TLR9 • => specialized in recognition of viral nucleicacids • Producehugequantities of Type I IFN (-a and –b) in response to virus MV ARNsb • 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 cellcarcinomawithAldara. 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. CD83 CD86 100 200 ** *** *** *** ** * 80 150 60 100 R-MFI % Positive Cells 20 40 20 0 0 IL3 IL3 MV MV R848 R848 MV+IL3 MV+IL3 M18 UV M18 UV M18 MV M18 MV CD40 A549 UV A549 UV A549 MV A549 MV Meso13 UV Meso13 UV Meso13 MV Meso13 MV 200 * 150 R-MFI 100 50 0 IL3 MV R848 MV+IL3 M18 UV M18 MV A549 UV A549 MV Meso13 UV Meso13 MV MV infected tumor cells induce maturation of plasmacytoid DC IL3 R848 MV IL-3 + MV Meso13 MV Meso13 UV

  11. MV infected tumor cells are internalized by plasmacytoid DC PKH67 PKH67 BDCA-4 HLA-DR Mo-DC pDC 4°C 37°C 4°C 37°C MV M18 UV MV HLA-DR Alexa568 PKH-67 MERGE A549 UV pDC + M18 MV pDC + A549 MV

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

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

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

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

  16. 20 0.6% 0.3% 15 + T CD8+ cells 10 g 5 % of IFN 0 79.6% 68% Mo-DCi LT only Mo-DCm + 0,1µM NY-ESO-1 Mo-DC + M18UV Mo-DC + M18MV 11.7% 8.15% 0.4% 0.16% NYESO-1 cross-presentation by pDC and Mo-DC exposed to MV infected tumor cells - Mo - DC pDC Mo DC Cross - presentation Ø NYESO - 1 [157 - 165] (10µM) pDC cross-presentation 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 Mo-DC + pep 1µM Mo-DC 0,5 95,1 % CD8+/IFNγ+ cells Mo-DC + IFN-γ + TNF-α Mo-DC + R848 Mo-DC 10,0 22,7 7,0 M18 MV (-) (-) (-) (-) R848 R848 R848 R848 Mo-DC IFN-γ TNF-a IFN-γ TNF-a IFN-γ TNF-a IFN-γ TNF-a M18 MV M18 UV M18 MV M18 UV 0,5 2,2 0,9 M18 UV (+) Mo-DC (-) Mo-DC (-) IFN-γ + TNF-α R848 1,2 4,9 1,3 Deauvieau F et al, Int J Cancer, 2014 M18 MV 0,5 2,3 0,7 M18 UV

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

  19. Funding Collaborations 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 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 Service de pneumologie du CHU de Lille Arnaud Scherpereel Institut Pasteur, Viral genomics and Vaccination laboratory Frédéric Tangy Mariana Mesel-Lemoine Centre de Recherche en cancérologie de Lyon Jenny Valladeau Christophe Caux

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