Repurposing the yellow fever vaccine for intratumoral immunotherapy
2019; Springer Nature; Volume: 12; Issue: 1 Linguagem: Inglês
10.15252/emmm.201910375
ISSN1757-4684
AutoresM. Ángela Aznar, Carmen Molina, Álvaro Teijeira, Inmaculada Rodríguez, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sánchez-Paulete, Luna Cordeiro, Iñaki Etxeberría, Maite Álvarez, Sergio Rius‐Rocabert, Estanislao Nistal‐Villán, Pedro Berraondo, Ignacio Melero,
Tópico(s)Viral Infections and Outbreaks Research
ResumoArticle19 November 2019Open Access Source DataTransparent process Repurposing the yellow fever vaccine for intratumoral immunotherapy Maria Angela Aznar Corresponding Author Maria Angela Aznar [email protected] orcid.org/0000-0002-7970-1672 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Carmen Molina Carmen Molina Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Alvaro Teijeira Alvaro Teijeira Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Inmaculada Rodriguez Inmaculada Rodriguez Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Arantza Azpilikueta Arantza Azpilikueta Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Saray Garasa Saray Garasa Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Alfonso R Sanchez-Paulete Alfonso R Sanchez-Paulete Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Luna Cordeiro Luna Cordeiro Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Iñaki Etxeberria Iñaki Etxeberria Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Maite Alvarez Maite Alvarez Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Sergio Rius-Rocabert Sergio Rius-Rocabert Microbiology Section, Dpto. CC, Farmaceuticas y de la Salud, Facultad de Farmacia, Universidad CEU San Pablo, CEU University, Boadilla del Monte, Madrid, Spain Instituto de Medicina Molecular Aplicada (IMMA), Universidad CEU San Pablo, Pablo-CEU, CEU Universities, Boadilla del Monte, Madrid, Spain Search for more papers by this author Estanislao Nistal-Villan Estanislao Nistal-Villan Microbiology Section, Dpto. CC, Farmaceuticas y de la Salud, Facultad de Farmacia, Universidad CEU San Pablo, CEU University, Boadilla del Monte, Madrid, Spain Instituto de Medicina Molecular Aplicada (IMMA), Universidad CEU San Pablo, Pablo-CEU, CEU Universities, Boadilla del Monte, Madrid, Spain Search for more papers by this author Pedro Berraondo Pedro Berraondo Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Ignacio Melero Corresponding Author Ignacio Melero [email protected] orcid.org/0000-0002-1360-348X Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Maria Angela Aznar Corresponding Author Maria Angela Aznar [email protected] orcid.org/0000-0002-7970-1672 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Carmen Molina Carmen Molina Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Alvaro Teijeira Alvaro Teijeira Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Inmaculada Rodriguez Inmaculada Rodriguez Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Arantza Azpilikueta Arantza Azpilikueta Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Saray Garasa Saray Garasa Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Alfonso R Sanchez-Paulete Alfonso R Sanchez-Paulete Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Luna Cordeiro Luna Cordeiro Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Iñaki Etxeberria Iñaki Etxeberria Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Maite Alvarez Maite Alvarez Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Search for more papers by this author Sergio Rius-Rocabert Sergio Rius-Rocabert Microbiology Section, Dpto. CC, Farmaceuticas y de la Salud, Facultad de Farmacia, Universidad CEU San Pablo, CEU University, Boadilla del Monte, Madrid, Spain Instituto de Medicina Molecular Aplicada (IMMA), Universidad CEU San Pablo, Pablo-CEU, CEU Universities, Boadilla del Monte, Madrid, Spain Search for more papers by this author Estanislao Nistal-Villan Estanislao Nistal-Villan Microbiology Section, Dpto. CC, Farmaceuticas y de la Salud, Facultad de Farmacia, Universidad CEU San Pablo, CEU University, Boadilla del Monte, Madrid, Spain Instituto de Medicina Molecular Aplicada (IMMA), Universidad CEU San Pablo, Pablo-CEU, CEU Universities, Boadilla del Monte, Madrid, Spain Search for more papers by this author Pedro Berraondo Pedro Berraondo Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Ignacio Melero Corresponding Author Ignacio Melero [email protected] orcid.org/0000-0002-1360-348X Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain CIBERONC, Madrid, Spain Instituto de investigación de Navarra (IDISNA), Pamplona, Spain Search for more papers by this author Author Information Maria Angela Aznar *,1,6, Carmen Molina1, Alvaro Teijeira1,2,3, Inmaculada Rodriguez1,2,3, Arantza Azpilikueta1,3, Saray Garasa1,3, Alfonso R Sanchez-Paulete1,7, Luna Cordeiro1,3, Iñaki Etxeberria1, Maite Alvarez1, Sergio Rius-Rocabert4,5, Estanislao Nistal-Villan4,5, Pedro Berraondo1,2,3 and Ignacio Melero *,1,2,3 1Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 2CIBERONC, Madrid, Spain 3Instituto de investigación de Navarra (IDISNA), Pamplona, Spain 4Microbiology Section, Dpto. CC, Farmaceuticas y de la Salud, Facultad de Farmacia, Universidad CEU San Pablo, CEU University, Boadilla del Monte, Madrid, Spain 5Instituto de Medicina Molecular Aplicada (IMMA), Universidad CEU San Pablo, Pablo-CEU, CEU Universities, Boadilla del Monte, Madrid, Spain 6Present address: Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 7Present address: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA *Corresponding author. Tel: +34 948194700; Fax: +34 948194717; E-mail: [email protected] *Corresponding author. Tel: +1 2155 734187; Fax: +34 948194717; E-mail: [email protected] EMBO Mol Med (2020)12:e10375https://doi.org/10.15252/emmm.201910375 See also: TC Wirth et al (January 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild-type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non-transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T-cell-dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T-cell infiltrates and a treatment-related reduction of Tregs. Additive efficacy effects were observed upon co-treatment with intratumoral 17D and systemic anti-CD137 and anti-PD-1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T-cell infiltration in the treated tumor. The repurposed use of a GMP-grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach. Synopsis The attenuated vaccine for yellow fever virus exerts antitumor effects when intratumorally injected which are mediated by immune responses and enhanced in pre-vaccinated mice. Repeated intratumoral injection of 17D virus stimulates antitumor responses and delays tumor growth in transplantable tumor models through a mechanism dependent on CD8+ T cells that requires cDC1 dendritic cells and functional IFNα signaling. 17D intratumoral injection modifies immune infiltration in injected and distant tumors as well as in tumor draining lymph nodes. Preimmunization of mice with 17D vaccine potentiates the antitumor effects of intratumoral 17D and this beneficial effect is transferred along with CD8 T cells. Introduction Intratumoral administration of immunotherapy agents is a strategy that aims to achieve better efficacy while minimizing systemic toxicity (Aznar et al, 2017; Marabelle et al, 2018). Engineered viruses selectively replicating in tumors (virotherapy) constitute an area of active development in which the therapeutic agents can be given systemically or locally (Turnbull et al, 2015; Bommareddy et al, 2018; Pol et al, 2018). In recent years, evidence has been accumulating that the ensuing antitumor cytolytic immune responses constitute the main mechanism of action of virotherapy rather than direct cytopathic effects (Bommareddy et al, 2018). As a result, immune-potentiating transgenes are usually cloned in the vectors to enhance efficacy (Hu et al, 2006; Kim et al, 2006; Zhang et al, 2011; Goins et al, 2014; Quetglas et al, 2015). Reminiscent of the intratumoral administration of live bacteria by William Cooley in the late 19th century (Coley, 1906, 1910), several groups have focused on the injection of replication-competent viruses into the tumor microenvironment of an injectable lesion seeking responses in distant metastases that are termed abscopal or anenestic effects (Aznar et al, 2017; Marabelle et al, 2018). In this regard, agents based on herpes simplex virus (HSV) (Goins et al, 2014), vaccinia virus (Kim et al, 2006), vesicular stomatitis virus (Patel et al, 2015; Kim et al, 2017), adenovirus (Jiang et al, 2017), new castle disease virus (NDV) (Lorence et al, 1994; Zamarin et al, 2014), and reovirus (Rajani et al, 2016; Samson et al, 2018) are the most advanced in their paths toward clinical use. The only FDA-approved agent to date is the HSV-based vector T-vec (Talimogene laherparepvec) because of its activity against unresectable or metastatic cutaneous melanoma (Andtbacka et al, 2015, 2016). In this case, the deletion-mutant attenuated virus is engineered to express GM-CSF and is used in patients preimmunized with the virus for safety reasons. Importantly, intratumoral T-vec seems to be clinically synergistic with the anti-PD-1 monoclonal antibody (mAb) pembrolizumab (Ribas et al, 2017), although confirmation is pending in an ongoing phase III clinical trial (NCT02263508). Moreover, clinical activity seems to be also enhanced by combination with the anti-CTLA-4 mAb ipilimumab (Puzanov et al, 2016). Reportedly, preimmunization to HSV does not hamper local injection therapy and is performed on purpose in the clinical setting as a safety measure. Evidence generated from intratumoral injection with wild-type NDV in mice indicates that most of the beneficial effects are immune-mediated by effector CD8 T cells and a synergistic combination with anti-CTLA-4 mAb was observed against tumor lesions not directly injected with virus (Zamarin et al, 2014). These results raise the question as to whether other viruses not affecting humans could also be used (Lizotte et al, 2015). However, veterinary or plant viruses are presumably not well adapted to human receptors and might be underperforming, even if they provide abundant nucleic acid with viral features acting as potent immune adjuvants on human pattern recognition receptors (PRRs). Interestingly in the case of NDV, it has been reported that preimmunization with virus not only does not hamper the therapeutic effects of local virotherapy but also actually enhances efficacy in mouse models (Ricca et al, 2018). Overall efficacy in virotherapy is probably the combined result of immunogenic cell death (Garg et al, 2017), ligands for pattern recognition receptors (Melero et al, 2015), and the ensuing immune response. Yellow fever is a serious infectious condition caused by a prototypic member of the Flavivirus genus and transmitted by Aedes Aegypti mosquito bites (Monath & Vasconcelos, 2015). Attenuation of this RNA(+) virus by serial passage (18 times in mouse embryo tissues, 58 times in minced whole chick embryo tissue, and 128 times in minced chick embryo without nervous tissue) (Lloyd et al, 1936) led to the development of the Nobel-laureated vaccine (Lemmel, 2001). The vaccine is fully sequenced, shows a good safety profile in immunocompetent adults, and is used prophylactically to prevent the disease in endemic regions and in travelers (World Health, 2017). Attenuation is related to a few nucleotide differences with the Asibi genome (encoding for 31 amino acid substitutions) and to reduction in the quasispecies diversity in the viral population (Hahn et al, 1987; Beck et al, 2014). Cancer treatment has been revolutionized by immunomodulatory monoclonal antibodies blocking the co-inhibitory receptor/ligand pair PD-1 and PD-L1 (Ribas & Wolchok, 2018). Moreover, strategies encompassing agonist monoclonal antibodies for costimulatory immune cell receptors such as CD137 (Morales-Kastresana et al, 2013) show potent effects against engrafted mouse tumors, are being clinically developed, and offer opportunities for synergistic combinations (Weigelin et al, 2015). We reasoned that cancer virotherapy could benefit from repurposing the use of an already approved and widely used live attenuated viral vaccine. In that regard, 17D yellow fever vaccine was considered an advantageous alternative, since most humans in Western countries are naïve to the natural pathogen or to the vaccine. Vaccination induces very potent cellular and humoral immunity resulting in strong and long-lasting CD8 T-cell memory and potently induces type I IFN (Gaucher et al, 2008; Bassi et al, 2015; Fuertes Marraco et al, 2015). In this study, we show that the yellow fever vaccine can be safely injected intratumorally in mice, giving rise to immune-mediated antitumor effects that can be combined with other immunotherapy agents in a clinically feasible fashion. Results 17D is cytopathogenic on an array of transplantable mouse and human cancer cell lines Infectivity of mouse and human tumors is considered a prerequisite for antitumor activity. Hence, we explored whether the 17D yellow fever virus vaccine strain (Stamaril) could kill a panel of tumor cell lines. Figure 1A shows that all mouse cell lines tested are susceptible to 17D infection, even though with different multiplicity of injection (MOI) requirements. Furthermore, similar data were obtained with a panel of human tumor cell lines representing colon cancer, renal cell carcinoma, breast cancer, and melanoma (Fig 1B), while non-transformed human fibroblasts were resistant to such cytopathic effect at the same range of MOIs (Fig 1C). Therefore, the 17D live attenuated strain is able to effectively infect and induce cell death of mouse and human tumor cell lines of different tissue origin at virus concentrations that are innocuous for non-transformed human cells. Interestingly, all murine cell lines tested are amenable to engraftment in mice for in vivo experimentation. Figure 1. 17D antitumor effects in vitro and in vivo A–C. The indicated mouse (n = 7) (A) and human (n = 7) (B) tumor cell lines, and non-transformed human fibroblasts (C) were exposed to increasing MOIs of 17D virus in culture, and subsequently cell viability was assessed by crystal violet staining performed 6 days later. The Vero cell line used for viral production is included as a positive control in each experiment with tumor cell lines, and the human tumor cell lines ARST1 and HCT 116 were included as positive controls of infection for the experiments with human non-transformed fibroblasts. Results shown are representative of at least three experiments similarly performed. D, E. MC38 tumors were engrafted and treated as schematically represented. Graphs represent individual tumor size follow-up upon intratumoral injections with 17D (n = 6) or vehicle (n = 6) as a control that are also shown as mean ± SD and as overall survival of the mice. Dashed lines indicate the injection days of 17D. ***P < 0.001. F, G. B16OVA-derived melanomas were engrafted and treated as schematically represented. Graphs represent individual tumor size follow-up upon intratumoral injections with 17D or vehicle as a control (n = 6 and n = 5 for 17D and control groups, respectively), also shown as mean ± SD and as overall survival of the mice. Dashed lines indicate the injection days of 17D. ***P < 0.001, **P < 0.01. H. Schematic representation of the experiments to engraft and treat MC38-derived bilateral subcutaneous tumors. I. Individual tumor growth follow-up and mean ± SD of tumor lesions directly injected with 17D or control vehicle (n = 6 per group). Dashed lines indicate the injection days in injected tumors. ***P < 0.001. J. Follow-up of contralateral non-injected tumors and mean ± SD of tumor lesions in the same mice as in (I). ***P < 0.001, *P < 0.05. Data information: Mean tumor volume growth over time was fitted using non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. Mantel–Cox test was used for survival analysis. Experiments are representative of at least two similarly performed. ***P < 0.001, **P < 0.01. Source data are available online for this figure. Source Data for Figure 1 [emmm201910375-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Intratumoral administration of 17D controls MC38 and B16OVA tumor progression MC38 and B16OVA cancer cell lines were among those susceptible to 17D infection in culture (Fig 1A). We next examined the effect of 17D upon repeated intratumoral administration into MC38 (Fig 1D and E) or B16-OVA (Fig 1F and G) established subcutaneous tumors. Although treatment was not curative in any case, a clear delay in tumor progression was observed. To exert such an effect, the 17D virus had to be competent since UV-inactivated viral particles failed to control tumor growth (Fig EV1A and B) when similarly injected. Click here to expand this figure. Figure EV1. Ultraviolet light inactivated 17D virus does not exert antitumor effects UV exposure inactivates 17D virus infectivity as observed in plaque-forming assays on Vero cells stained with crystal violet. Comparative antitumor effect against established MC38-derived tumors intratumorally injected with replication-competent and UV-inactivated 17D virus as indicated. Individual tumor growth follow-up and mean ± SD of tumor lesions are shown, (n = 5 per group). Mean tumor volume growth over time was fitted using non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. ***P < 0.001. Download figure Download PowerPoint Having proven a reproducible control of tumor growth following intratumoral administration of 17D, we addressed the issue of whether a contralateral tumor (not directly injected) could be controlled as well in bilateral MC38 tumor models (Fig 1H–J). Experiments in Fig 1J show that certain contralateral therapeutic effects could be observed, although this statistical difference was lost at later time points (Fig 1I). Notably, such efficacy could not be attributed to the direct viral infection of the non-injected tumor, since 17D nucleic acid sequences were not detected in distant tumors of 17D-treated mice by a sensitive quantitative RT–PCR assay (Fig EV2A and B). Click here to expand this figure. Figure EV2. Lack of 17D virus detection in contralateral tumors Schematic representation of the experiment in mice bearing bilateral MC38 tumors that were excised on day +12 (2 days following a second dose of intratumoral 17D virus or control vehicle, n = 9 per group). Quantitative RT–PCR detection of 17D in the indicated directly injected or contralateral tumors. Individual cases are represented and the median indicated by a horizontal line. Download figure Download PowerPoint 17D therapeutic effects are mediated by CD8 T cells Next, we examined the necessary contribution of the different lymphocyte subsets by selective depletion of CD4 and CD8 T cells in bilateral MC38 tumor-bearing mice. Experiments shown in Fig 2A demonstrate that that CD8 T-cell depletion with an anti-CD8β mAb completely abolished 17D-induced tumor control in the directly injected (Fig 2B) and in the contralateral tumor nodules (Fig 2C), and lead to reduced overall survival (Fig 2D). In the case of CD4 depletion, no effect was seen in the treated tumor, but the absence of CD4 T cells improved the therapeutic effects in the contralateral tumor. Experiments in Fig EV3A–C depleting granulocytes and myeloid suppressor cells or NK/NKT cells yielded negative results, indicating that these leukocyte subsets do not have a necessary role in the therapeutic effects of 17D intratumoral injections. Selective leukocyte depletions were confirmed in every experiment in peripheral blood (Fig EV3D). Figure 2. T-cell requirement for the antitumor effects of 17D intratumoral injections Schematic representation of the experiments upon treatment of mice bearing bilateral MC-38-derived colon carcinomas when concurrently eliminating CD4 or CD8 T cells with depleting monoclonal antibodies as indicated. Individual follow-up and mean ± SD (n = 6 per group) of tumor sizes in the directly 17D or control vehicle-injected tumors. Dashed lines indicate the days of intratumoral injection. Individual tumor growth and mean ± SD follow-up of the size of contralateral (non-directly injected) tumors under the same indicated conditions (n = 6 per group). Overall survival of the indicated groups of mice (n = 6 per group). Data information: Mean tumor volume growth over time was fitted using non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. Mantel–Cox test was used for survival analysis. ***P < 0.001, **P < 0.01, *P < 0.05, ns: non-significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. NK1.1 and GR-1 depletions and validation of selective subset depletion experiments A. Scheme of depletion experiments as in Fig 4 but depleting NK1.1+ cells (NK and NKT cells) or GR1+ cells (MDSC and granulocytes). B, C. Tumor size follow-up of the indicated experimental groups (n = 6 per group) measuring directly 17D virus-injected (B) and contralateral untreated tumors (C), respectively. D. Verification by flow cytometry of cell depletions in peripheral blood in experiments (mean ± SD, n = 4 per group) from Figs 4 and EV3. Data information: Mean tumor volume growth over time was fitted using non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. Unpaired T test was used to calculate depletion efficiency. **P < 0.01***P < 0.001. Download figure Download PowerPoint Intratumoral 17D used in combination with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies Given the partial efficacy of the intratumoral 17D-mediated by CD8 T-cell responses, we investigated if this treatment strategy could be enhanced by mAbs acting on costimulatory or co-inhibitory pathways (Melero et al, 2007). Setting up bilateral MC38-derived tumor nodules, we tested combinations of repeated intratumoral 17D and systemic anti-CD137 or anti-PD-1 mAbs (Fig 3A). Very clear synergistic tumor-eradicating responses were observed in directly injected tumors when 17D is combined with systemic anti-CD137 mAb (Melero et al, 1997) that was almost ineffective by itself (Fig 3B). Anti-PD-1 mAb, also ineffective by itself, showed mild local additive efficacy when used in combination with intratumoral 17D regimen (Fig 3B). Regarding contralateral efficacy, a certain delay in tumor growth was achieved in mice contralaterally treated with 17D and systemic anti-PD-1 mAb. Systemic anti-CD137 by itself delayed contralateral tumor growth to a similar degree without further improvement by 17D treatment of the contralateral tumor (Fig 3C). This leads to an improved overall survival of the mouse group treated with 17D+ anti-CD137 (Fig 3D). These observations, especially the efficacy achieved in 17D-injected tumors, suggest the clinical interest of anti-CD137-targeted agonists (Compte et al, 2018) to be used in conjunction with intratumoral 17D. Figure 3. Combinations of intratumoral 17D virotherapy and systemic administration of anti-PD-1 and anti-CD137 immunomodulatory monoclonal antibodies Schematic representation of the experiments performed in mice bearing bilaterally MC38-derived tumors treated on the indicated days with intratumoral 17D virus or control vehicle in one of the lesions and intraperitoneally with the indicated immunotherapeutic monoclonal antibodies anti-PD-1, anti-CD137, or control RIgG. Individual and averaged (mean ± SD) tumor size progression of the indicated experimental groups. Dashed lines indicate the days of intratumoral injection. Individual and averaged (mean ± SD) tumor size follow-up of the contralateral tumors that were not intratumorally injected. Overall survival of the indicated groups of mice. Data information: n = 8 mice/group in all the groups except for Control + anti-PD-1 and Control + anti-CD137 (n = 7 mice/group). Mean tumor volume growth over time was fitted using non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. Mantel–Cox test was used for survival analysis. Data are representative of two experiments identically performed. ***P < 0.001, **P < 0.01, *P < 0.05, ns: non-significant. Download figure Download PowerPoint Immune events underlying intratumoral 17D therapeutic effects CD8-mediated antitumor responses are completely dependent on conventional type I dendritic cells (cDC1) (Hildner et al, 2008; Murphy et al, 2016; Sanchez-Paulete et al, 2017). Such antigen-presenting cells that mediate CD8 T-cell crosspriming (Sanchez-Paulete et al, 2017) of tumor antigens are defective in BATF3−/− mice showing that they are required for a number of successful immunotherapies to work (Salmon et al, 2016; Sanchez-Paulete et al, 2016; Spranger et al, 2017). Consistent with this notion, the tumor growth delay mediated by intratumoral 17D injection was lost in BATF3−/− mice (Fig 4A–C), especially in non-injected contralateral tumors (Fig 4C). Figure 4. Treatment efficacy reduction in BATF-3 knockout mice A. Scheme representing experiments as in Fig 2 performed in WT and BATF-3−/− mice bearing bilateral MC38 tumors and treated as indicated. B, C. Size follow-up of treated and contralateral tumors of the indicated groups of mice (mean ± SD), (n = 5 for all 17D-injected groups, WT Control + RIgG n = 6, WT Control + anti-PD-1 and WT Control + anti-CD137 n = 7, KO Control + RIgG and KO Control + anti-CD137 n = 6, and KO Control + anti-PD-1 n = 7). Mean tumor volume growth differences were calculated with non-linear regression curve fit. Treatments were compared using the extra sum-of-squares F-test. ***P < 0.001,**P < 0.01, *P < 0.05, ns: non-significant. Source data are available online for this figure. Source Data for Figure 4 [emmm201910375-sup-0004-SDataFig4.xlsx] Download figure Download PowerPoint We have previously reported that antitumor immunotherapy elicited by anti-CD137 and anti-PD-1 mAbs was totally dependent on the performance of cDC1 cells in tumor antigen crosspriming (Sanchez-Paulete et al, 2016). In keeping with these findings, the local synergisti
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