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 Infection apoptosis Activation of the immune system Oncolytic virus Infection Tumor cells Healthy cells Ideal replicating oncolytic virus: (Russell SJ, Nature biotechnol, 2012 Vacchelli E et al, Oncoimmunology, 2013) - Infects exclusively or preferentially tumor 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.
Measles virus vaccine () based anti-tumoral virotherapy Hémagglutinine (H) Protéine de Fusion (F) Protéine de Matrice (M) Protéine L Phosphoprotéine (P) ARN + Nucléoprotéine (N) Double membrane lipidique Guillerme JB et al, Biology, 2013 Measles virus vaccine, Schwarz strain (): 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 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)
infection of Malignant Pleural Mesothelioma tumor cells Video : infection of meso13 by -egfp (enhanced green fluorescent protein)
Limited infection of healthy mesothelial cells Video : healthy mesothelial cells exposed to -egfp
Activation of the immune system by infected tumor cells? Tumor cells Apoptosis Danger signals DAMPs? PAMPs? Immature DC Cross presentation to Mature DC? CD8+ T Lymphocytes (CTL)? Myeloid DC? Plasmacytoid DC?
Activation of monocyte derived DC (Mo-DC) and tumor antigen cross-priming by infected tumor cells Mo-DC activation Tumor antigen cross-priming 80 CD83 IL-12p70 10000 60 40 20 5000 % CD83 + cells IL-12p70 (pg/ml) 20 10 CD86 IL-10 3000 2000 1000 Median Fluorescence Intensity IL-10 (pg/ml) IFN-α DCi / Poly I:C alone Meso13 M6 IFN-α DCi / Poly I:C alone Meso13 M6 Gauvrit et al, Cancer Research, 2008
Plasmacytoid Dendritic Cells (pdc) Expression of TLR7 et TLR9 => specialized in recognition of viral nucleic acids ARNsb Producehugequantitiesof Type I IFN (-αand β) 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) Antigencross presentationin mice: Cross-tolérance(Goubier, A., Dubois, B., Immunity, 2008) OVA (Mouries, J., Blood, 2008) One of the targetof imiquimod(r837, TLR7 ligand) in the treatment of basal cell carcinoma with Aldara. IFN-α Production and tumor antigen cross-presentation by pdc exposed to infected tumor cells?
pdc are not infected by Schwarz Video : pdc exposed to -egfp
infected tumor cells induce maturation of plasmacytoid DC FSC BDCA4 CD83 CD86 CD40 CD83 CD86 9 % 92.4 % 4,27 130,96 24 167 20 % 5,13 10 SSC CD123 IL3 +IL3 M18 M18 UV Meso13 Meso13 UV A549 A549 UV IL3 +IL3 M18 M18 UV IL3 IL-3 + 100 80 60 40 ** *** *** ** *** % Positive Cells Meso13 Meso13 UV A549 A549 UV 200 150 20 100 * R-MFI Meso13 Meso13 UV 20 0 0 92.4 % 5,99 60 CD40 200 * 150 90.6 % 12,36 85 100 50 7.42 % 5,33 22 0 IL3 +IL3 M18 M18 UV Meso13 Meso13 UV A549 A549 UV R-MFI
infected tumor cells are internalized by plasmacytoid DC Mo-DC pdc 4 C 37 C 4 C 37 C HLA-DR Alexa568 PKH-67 MERGE M18 UV pdc + M18 A549 pdc + A549 UV PKH67 PKH67 HLA-DR BDCA-4
activates pdc IFN-α production by TLR7 600 800 IFN-α (ng/ml) 400 200 30 20 10 0 IL3 IL3 + M18 M18 UV A549 A549 UV pd DC IFN-α(ng/ml) α 600 400 200 80 60 40 20 0 IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1 IRS661 IRS661 IRS661 IL-3 + *10 CpG-A M18 (TLR-9) IRS661: TLR7inhibitor
probably activates pdc via TLR7 in early endosome CpG-A (TLR9) CpG-B (TLR9) -> High IFN-α -> Low IFN-α -> Low costimulation (CD80, CD86) -> High costimulation (CD80, CD86) Kerkmann M et al, J Immunol, 2003 -> Low inflammatory molecules secretion (IL-8) -> High inflammatory molecules secretion (IL-8) Early endosome Late endosome Honda K et al, Nature, 2005 Guiducci C et al, J Exp Med, 2006 IRF7 pathway NF-κB pathway Sadaka C et al, Blood, 2009 Kubo-Murai M et al, Blood, 2008 HIV (TLR7) (TLR7/TLR8) CpG-B (TLR9) O brien M et al, J Clin Invest, 2011 (TLR7) (TLR7/TLR8) Guillerme JB et al, clin cancer Res, 2013
NYESO-1 cross-presentation by pdc exposed to infected tumor cells NYESO-1 (NYESO-1 / RPLPO)*1000 15000 10000 5000 0 M 18 A549 NI UV 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 infected tumor cells LT only pdc+ LT 0.1% 0.1% CD8 CD8 IFN-γ IFN-γ LT + pdc 0.1µM NYESO-1 [157-165] 1µM NYESO-1 [157-165] 10.8% 87.5% UV 6.5% 0.2% M18 0.8% 0.2% A549 20 10 0 % of IFNγ+ T CD8+ cells IFN-γ IFN-γ LT - pdc M18 M18 UV 0.3% 0.1% CD8 CD8 CD8 CD8 LT only (n=5) pdc + LT (n=6) pdc 0.1 µm NYESO-1 (n=3) pdc + M18 (n=6) pdc + M18 UV(n=5) IFN-γ IFN-γ
NYESO-1 cross-presentation by pdc and Mo-DC exposed to infected tumor cells Ø NYESO-1 [157-165] (10µM) Mo-DC pdc 0.6% 68% 0.3% 79.6% 20 15 10 5 0 Mo - DC Cross- presentation LT only Mo-DCi Mo-DCm + 0,1µM NY-ESO-1 Mo-DC + M18 Mo-DC + M18UV % of IFN γ + T CD8+ cells 11.7% M18 HLA-A*0201 neg /NY-ESO-1 pos 0.16% M18 UV HLA-A*0201 neg /NY-ESO-1 pos CD8 CD8 IFN-y IFN-y 8.15% 0.4% 20 pdc cross-presentation 10 0 LT only (n=5) pdc + LT (n=6) pdc 0.1 µm NYESO-1 (n=3) pdc + M18 (n=6) pdc + M18 UV(n=5) % of IFNγ+ T CD8+ cells
NYESO-1 cross-presentation by Mo-DC is increased by TNF-α/IFN-γ Mo-DC Mo-DC 0,5 Mo-DC + pep 1µM 95,1 Mo-DC + IFN-γ + TNF-α Mo-DC + % CD8 + /IFNγ + cells 30 20 10 M18 M18 UV 10,0 22,7 7,0 0 0,5 2,2 0,9 Mo-DC (-) IF FN-γ TNF-α (-) IF FN-γ TNF-α (-) IF FN-γ TNF-α (-) IF FN-γ TNF-α M18 M18 UV M18 M18 UV (+) Mo-DC (-) Mo-DC M18 (-) IFN-γ+ TNF-α 1,2 4,9 1,3 Deauvieau F et al, Int J Cancer, 2014 M18 UV 0,5 2,3 0,7
Conclusions Blood myeloid DC? CD1c+ DC? CD141+ DC? Gauvrit A et al, Cancer Research, 2008 Prefered Infection Limited infection Tumor cells (CD46 high ) Healthy cells (CD46 low ) Apoptosis -infected Tumor cells Phagocytosis Maturation ->Cross-priming of TAA-specific CD8+ T cell response TAA PAMP? DAMP (HSP70, gp96) (IFN-α, IFN-β, HMGB1, IL-6 IL-8) Phagocytosis Immature Mo-DC TAA PAMP ( Single-strand RNA) DAMP? Immature plasmacytoid DC Maturation Mature Mo-DC Mature plasmacytoid DC ->Cross-presentation of TAA to specific CD8+ T cells Cross-priming? ->Production of a large quantity of IFN-α Guillerme JB et al, Clinical Cancer Research, 2013 Donnelly OG et al, Gene Ther, 2013
Funding Collaborations 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 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 Institut Pasteur, Viral genomics and Vaccination laboratory Frédéric Tangy Mariana Mesel-Lemoine Centre de Recherche en cancérologie de Lyon Jenny Valladeau Christophe Caux