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-44, France. Pasteur Institute, Viral Genomics and Vaccination, Paris, 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, 212 Vacchelli E et al, Oncoimmunology, 213 - 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, 213 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 CD15/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, 28; Boisgerault N et al, Biomed Res Int, 213
Oncovirotherapy Oncolytic properties of Measles Vaccine Tumour Cell Oncolytic properties Immunogenic apoptotic cell death Virus Oncolytic (Vaccine strain) immunogenic molecules DAMP & PAMP Specific T cell response T Lymphocytes Myéloïd DC Plasmacytoïd DC
% egfp + cells Tumour cell infection (mesothelioma) INFECTION Mesothelioma Infected cells -egfp Others Mesothelioma : 14/18 Melanoma : 7/8 Lung : 3/4 Colorectal : 4/4 1 8 6 4 2 Meso11 Meso13 Meso47 Meso56 Meso96 24 48 72 Time post infection (h) Syncitium Mesothelioma (Gauvrit A et al, Cancer Research, 28) Lung, Colorectal adenocarcinoma (N. Boisgerault et al., Biomed. Res. Int., 213)
Normal cells: mesothelial and lung cells Mesothelial cells Lung cells Also for : Fibroblasts Endothelial cells
Oncovirotherapy - Live attenuated Schwarz Measles Vaccine Tumour Cell Oncolytic properties Immunogenic apoptotic cell death Virus Oncolytic (Vaccine strain) immunogenic molecules DAMP & PAMP Specific T cell response T Lymphocytes Myéloïd DC Plasmacytoïd DC
Median Fluorescence Intensity HMGB1 (ng/ml) % Calreticulin + cells Danger Signals: Damage Associated Molecular Patterns Bianchi and Manfredi, 27 Kepp et al., 29 HSP7 Cross-presentation of tumor-derived antigens HMGB1 Antigen processing and presentation Calreticulin Phagocytosis 4 5 2 Control 3 1 2 1 25 Meso13 A549 M6 HT29 Meso13 A549 M6 HT29 Meso13 ADK153 M6 HT29 Gregoire et al, in preparation
Immune response with Live attenuated Schwarz Measles Vaccine Tumour Cell Oncolytic properties Immunogenic apoptotic cell death Virus Oncolytic (Vaccine strain) immunogenic molecules DAMP & PAMP Specific T cell response Activation maturation T Lymphocytes Myéloïd DC Plasmacytoïd DC
preserves dendritic cells Mo-DC pdc Myeloid dendritic cells (Gauvrit A et al., 28, Cancer Research) Plasmacytoid dendritic cells (Guillerme JB et al., 213, Clinical Cancer Research)
-infected tumor cells induced Mo-DC Maturation MFI MFI IFNα - PolyI:C TNFα - PolyI:C MFI MFI IFNα - PolyI:C TNFα - PolyI:C MFI % CD83 + cells IFNα - PolyI:C TNFα - PolyI:C 2 CMH I HLA-A,B,C 2 CD8 CD8 1 CD83 CD83 1 1 5 1 Dci IFN TNF M11 UV HLA-DR M11 CMH II M13 UV M13 6 Dci IFN TNF M11 UV M11 CD86 CD86 M13 UV M13 1 Dci IFN TNF M11 UV M11 CD4 CD4 M13 UV M13 8 5 4 2 6 4 2 DCi Dci IFN TNF UV M11 UV M11 UV M13 UV M13 Meso 11 Meso 13 DCi Dci IFN TNF UV M11 UV M11 UV M13 UV M13 Meso 11 Meso 13 DCi Dci IFN TNF UV M11 UV M11 UV M13 UV M13 Meso 11 Meso 13 (Gauvrit A et al., 28, Cancer Research)
% CD8+ IFN- + cpm cpm MUC-1 or mesothelin +Mo-DC 1 T CD4 Proliferation CD4 4 T CD8 Proliferation CD8 3 5 2 1 DCi IFN -PolyI:C UV DCi IFN -PolyI:C UV Meso 13 Meso 13 Clone T CD8 anti-muc-1 Cross-presentation (mesothelin) 1 5 DCi TNFα- PolyI:C (Gauvrit A et al., 28, Cancer Research) Meso 13 (Gauvrit et al., 28)
Plasmacytoid dendritic cells Expression of TLR7 et TLR9 => specialized in recognition of viral nucleic acids ARNsb Produce huge quantities of Type I IFN (- and b) in response to virus Antigen cross presentation in human: HIV (Hoeffel, G,.Immunity, 27) (Crozat, K., J Exp Med, 21) Influenza (Lui, G., PLoS One, 29) Antigen cross presentation in mice: Cross-tolérance (Goubier, A., Dubois, B., Immunity, 28) OVA (Mouries, J., Blood, 28) One of the target of imiquimod (R837, TLR7 ligand) in the treatment of basal cell carcinoma with Aldara. IFN- Production and tumor antigen cross-presentation by pdc exposed to infected tumor cells?
PKH67 PKH67 infected tumor cells are internalized by Plasmacytoid dendritic cells 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 HLA-DR BDCA-4
IL3 +IL3 R848 M18 M18 UV Meso13 Meso13 UV A549 A549 UV R-MFI SSC CD123 IL3 +IL3 R848 M18 M18 UV Meso13 Meso13 UV A549 A549 UV IL3 +IL3 R848 M18 M18 UV Meso13 Meso13 UV A549 A549 UV % Positive Cells R-MFI infected tumor cells induce Plasmacytoid dendritic cells maturation FSC BDCA4 CD83 CD86 CD4 CD83 CD86 IL3 9 % 4,27 24 1 8 *** ** *** ** *** 2 15 * R848 92.4 % 13,96 167 6 4 1 2 2 2 % 5,13 1 IL-3 + 92.4 % 5,99 6 2 * CD4 15 Meso13 9.6 % 12,36 85 1 5 Meso13 UV 7.42 % 5,33 22
IL3 IL3 + R848 M18 M18 UV A549 A549 UV IFN- (ng/ml) pdc IFN-α (ng/ml) IFN- (ng/ml) infected tumor cells induce production of INF type I by pdc 6 4 2 3 2 1 8 6 4 2 8 6 4 2 IL-3 IL-3,1,1,5,1,5,1,5 1,5 1,1,1,5,5 1,1,1,5,5 1 IRS661 IRS661 IRS661 IRS661: TLR7inhibitor IL-3 IL-3 + *1 *1 CpG-A CpG-A M18 CpG-A CpG-A (TLR-9)
CD8 CD8 CD8 CD8 LT only (n=5) pdc + LT (n=6) pdc.1 µm NYESO-1 (n=3) pdc + M18 (n=6) pdc + M18 UV(n=5) % of IFN + T CD8+ cells CD8 CD8 infected tumor cells induce NY-ESO-1 cross-presentation by pdc LT only pdc+ LT.1%.1% IFN-γ IFN-γ LT + pdc.1µm NYESO-1 [157-165] 1µM NYESO-1 [157-165] 1.8% 87.5% R848 2 6.5% UV.2% 1 M18.8%.2% A549 IFN-γ LT - pdc IFN-γ M18 M18 UV.3%.1% IFN-γ IFN-γ
In conclusion: Virotherapy and DC activations Tumor cells Apoptosis Dendritic cells Cytotoxic CD8+ T Lymphocytes (CTL) Dangers signal expression HMGB1 Calreticuline HSP inflammasome, caspase1, IL-1b Boisgerault N et al, in preparation Immune system activation Activation of myeloïd DC Activation of tumor antigen specific CD8+ T lymphocytes Gauvrit A et al, Cancer Research, 28 Immune system activation Activation of plasmacytoid DC (production of type I interferon) Guillerme JB et al, Clin. Cancer Res., 213
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