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Abscopal effect seen with T-cell therapy enhancing oncolytic adenoviruses armed with TNFα and IL-2 Riikka Havunen1, Suvi Parviainen1,4, Mikko Siurala1,4, Michael Behr1, Dirk Nettelbeck2, Anja Ehrhardt3, Akseli Hemminki1,4 1Cancer Gene Therapy Group, Medicum, Haartman Institute, University of Helsinki, Helsinki, Finland 2German Cancer Research Center (DKFZ), Heidelberg, Germany 3Institute for Virology and Microbiology, University Witten/Herdecke, Witten, Germany 4TILT Biotherapeutics Ltd, Helsinki, Finland Background Interleukin (IL) -2 and tumor necrosis factor alpha (TNFa) are promising for enhancing antitumor effects of adenoviruses. Virotherapy can be further boosted with Tumor-Infiltrating Lymphocyte (TIL) therapy With armed viruses, effective and safe cytokine expression can be achieved locally instead of high systemic level administration. Aims To study abscopal effect: Is localized treatment of a tumor with non-oncolytic adenoviruses carrying murine cytokines (Ad5-CMV-mTNFa/mIL2) able to affect also distant tumor? To construct and characterize oncolytic adenoviruses coding for human IL-2 and TNFa (Ad5/3-E2F-d24-hTNFa-IRES-hIL2) Results Oncolytic adenoviruses with human IL-2 and TNFa kill tumor cells and produce active cytokines in vitro. a. OVCAR3 - Ovarian cancer 120 120 100 100 Viability (%) Viability (%) A549 - Lung cancer 80 60 40 20 80 60 40 20 0 0 0 1 10 100 1000 0 1 10 VP/cell 120 120 100 100 Viability (%) Viability (%) Panc1 - Pancreatic cancer 80 60 40 20 80 60 40 20 0 0 0 1 10 100 1000 0 1 10 VP/cell The abscopal effect was seen: Treatment of a tumor with adenoviruses also reduced the growth of untreated tumors. Treatment influences distant tumors by activating immune responses and not by spreading the virus. Virus treatment recruits at least Natural Killer cells to both tumors and reduces the presence of immunosuppressive M2 macrophages. Oncolytic adenoviruses with human cytokines are effective in vitro. IL2 1:100 TNFa+IL2 TNFa 1:4000 Viability (%) 120 100 80 60 40 20 0 TNFa+IL2 mock d. IL-2 activity Mock Conclusions 1 0,8 0,6 0,4 0,2 0 c. hIL2 10 ng/ml ng/ml IL-2 and TNFa expression by the infected cells 100 1000 VP/cell Viability (%) b. 1000 VP/cell SK-MEL-28 - Melanoma Figure 3: Mice were sacrificed on day 8. Tumor samples were analyzed with FACS to detect Natural killer cells, M2 macrophages, dentritic cells and CD8+ T cells with melanoma-specific markers. Changes in CD8+ cells were undetectable. Means +/- SEM are shown. Ns=not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 100 TNFa activity 120 100 80 60 40 20 0 TNFa+IL2 Virus injections recruit Natural Killer cells to both tumors and treatment with cytokine-armed viruses reduces the presence of immunosuppressive M2 macrophages. Figure 2: Viral DNA was explored from both injected and non-injected tumors with qPCR. Median amount of viral DNA normalized against murine β-actin is shown. hTNFa 10 ng/ml Figure 1: Two B16-OVA murine melanoma tumors were implanted in C57BL/6 mice. One of the two tumors was treated intratumorally with cytokine- or Luc1-bearing adenoviruses (10e9 VP/tumor) on days 1 and 4. OT-1 T cells were administrated intraperitoneally (1.5x10e6 ) on day 1. Tumor growth was followed for 8 days; mock is set as 100%. Mean + SEM are shown. *p<0.05; ***p<0.001; ****p<0.0001 The virus does not spread to distant tumors. Mock Viral treatment induces growth suppression not only at the injection site but also at distant tumor. Figure 4 : Cancer cell lines were infected with Ad5/3-E2F-d24hTNFa-IRES-hIL2 or controls. (a) Cell survival was measured after 3 to 8 days. (b) Expression of cytokines was measured with FACSbased bead array and activity (c and d) with indicator cell lines. Means + StDev are shown. VP=viral particles