Poster Session: Combination Therapy

3rd Immunotherapy of Cancer Conference / European Journal of Cancer 55S1 (2016) S1–S30

Introduction: Immunotherapy has emerged in recent years as a new strategy to treat various kinds of diseases. Programmed Cell Death Ligand 1 (PD-L1) is a membrane molecule involved in different proliferation signalling pathways. This molecule engages PD-1, expressed in T cells, which transmits an inhibitory signal that maintains self-tolerance. However, cancer uses this pathway to suppress tumour immunity and proliferate. Monoclonal antibodies (mAbs) against PD-1 and PD-L1, two relevant immune checkpoints, is the new therapy to promote antitumor efficacy. These mAbs can be combined with other therapeutic agents as Doxorubicin (Dox). Its immunogenic properties contribute to enhance cell death, and then, the antitumor efficacy. However, its toxicity has been controlled by its encapsulation in different liposomal formulations. Therefore, the aim of this project was the development of PD-L1 targeted Dox immunoliposomes and its pharmacokinetic/pharmacodynamic evaluation in an in vitro/in vivo platform using a melanoma cell line. Materials and methods: Dox liposomes were prepared by the filmhydration method, combined with a pH gradient. Briefly, lipids were dissolved in a chloroform:methanol solution. The film obtained was hydrated with ammonium sulphate buffer. The liposomal solution was extruded to obtain a homogeneous population. They were washed and incubated with Dox, forming LPDOX. PD-L1 targeted Dox liposomes (LPDOXFab’) were formulated according to the post-insertion method. Micelles were prepared in and incubated with anti-PD-L1-Fab’ fragments. Afterwards, Fab’ conjugated micelles were incubated with LPDOX. LPDOXFab’ was purified by centrifugation. Particle size, PDI and Zeta potential were analyzed. Dox EE and coupling efficiency were quantified. Drug release profile was measured at pH 7.4 for 24h. For in vitro/in vivo studies, B16 OVA melanoma cell line was used. Cytotoxicity (IC50) was obtained after 24 h exposure. Dox cell incorporation was measured after 4 h. Currently, a melanoma mouse model is being used to assay antitumor efficacy. Results and Discussion: The post-insertion method allowed us to develop PD-L1 targeted Dox liposomes. Particle size was approx. 157±1.8 nm associated with a low PDI. The EE was 95±7.5% and ligand conjugation was 40±7.4%. Drug release for both types of formulations reached 35%. Dox reached the highest cytotoxicity followed by LPDOX and LPDOXFab’, which did not differ statistically, supporting the similar Dox intracellular accumulation.In vivo experiments are being carried out by our group. Conclusions: PD-L1 targeted Dox liposomes have been successfully developed by the post-insertion method. In vitro studies revealed similar behaviour of targeted and non-targeted formulations. However, the role of the immune response is being assayed in an in vivo model, as well as the blocking mechanism of the anti-PD-L1-Fab’. Conflict of interest: No conflict of interest.

54 Changes in plasma HMGB1 concentration during conventional treatment of lung adenocarcinoma patients are associated with overall survival D. Aguilar-Cazares1, M. Meneses-Flores1, C. Camacho-Mendoza1, H. Prado-Garcia1, M. Galicia-Velasco1, J.S. Lopez-Gonzalez1 1 Instituto Nacional de Enfermedades Respiratorias, Laboratorio de Cancer Pulmonar, Distrito Federal, Mexico Background: The more prevalent histological subtype of non-small cell lung carcinoma is the adenocarcinoma. Treatment for this histological subtype with no EGFR mutations remains in platinum-based chemotherapy. High mobility group box 1 (HMGB1) is an essential activator of cellular response to genotoxicity induced by cisplatin. HMGB1 plays a dual role, some reports indicates that HMGB1 binds to cisplatin-DNA-adduct favoring DNA repair and chemoresistance. In contrast, other reports show that cell death induced by platinum-based compounds releases HMGB1 for stimulating the anti-tumor immune response.

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Objectives: In the cohort of lung adenocarcinoma patients, plasma HMGB1 concentrations previous and during treatment were quantified and fluctuations were associated with overall survival. Materials and methods: HMGB1 in plasma of 100 lung adenocarcinoma patients was quantified by ELISA and was associated with overall survival. Results: In cisplatin-treated patients, according to HMGB1 plasma concentrations, four distinct behavioral profiles were detected. The patient group that initially increase but after decrease HMGB1 was associated with poor overall survival (os) (median os = 10 months) compare to other groups. According to overall survival, a significance difference between the group with poor overall survival and the group that always increase HMGB1 during cisplatin treatment (median os = 15 months) were found (log rank test p-value = 0.006). The data suggest that, during conventional lung cancer treatment, level of HMGB1 associate to overall survival. Conclusion: These findings suggest that in addition to clinical and radiological evaluations in lung adenocarcinoma patients, HMGB1 quantification might be translated into clinical practice as a biomarker associated with overall survival. Conflict of interest: No conflict of interest.

Poster Session: Combination Therapy

55 Strategy for synchronous and multiple liver metastasis S. Osada1 1 Gifu University School of Medicine, Multidisciplinary Therapy for Hepato-Biliary-Pancreatic Cancer, Gifu, Japan Background: Surgical indications for resection of synchronous meta­ stasis from colorectal cancer (CRC) and the optimal timing of hepatectomy are still controversial and widely debated. Patients and methods: Synchronous and multiple metastatic liver tumors were detected in 57 since May/2005. Our treatment policy has been to perform hepatectomy first, if the resection can be done with no limit on size and number of tumors. However, if curative resection is not, chemotherapy is begun first and timing for the possibility of a radical operation is planned immediately. Results: 1) In 37 patients whose tumors were located only in the liver, primary tumor resection was performed first in 16 patients, and after tumordecreasing by chemotherapy, operation was performed in 7 patients. In 20 patients in whom chemotherapy was performed first, after controlling the distant metastasis, hepatectomy was performed in 3 patients, and staged hepatectomy was performed in 10 patients. 2) Recurrence was detected after hepatectomy in 75.0% of simultaneous resection cases and in 70.0% of staged cases. In the recurrence cases, early detection (within 6 months) after tumor resection occurred in 58.3% of the simultaneous and 14.2% of the staged. 3) No differences in results of pre- and postoperative liver function tests were found between these groups, and duration of hepatectomy and blood loss were also similar. No deaths occurred, and one incidence of bile leakage was detected in each group. 4) Median survival time (MST) and 2-year survival rate were significantly better in the hepatic resection cases than in the non-operated cases. There was no significant difference in MST or 2-year survival rate between simultaneous and staged cases. 5) In 10 staged cases, length of chemotherapy had no effect on pre- or postoperative liver function test results, and survival curves. 6) Repeat operation was performed for recurrence in 75% of the simultaneous and 14.3% of the staged cases. The average time between first and second operation was 13.1±7.7 months, and 2-year survival was 100%. Conclusion: Neoadjuvant chemotherapy does not increase the risk of postoperative complications or the surgical difficulties of hepatectomy for colorectal metastases. Treatment strategies for these clinical conditions should include consideration of responsible administration of chemotherapy and surgery.

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Conflict of interest: No conflict of interest.

56 Tumor cell death induced by modulated electrohyperthermia in combination with Marsdenia tenacissima in murine colorectal allograft tumor model N. Meggyeshazi1, C. Kovago2, T. Vancsik1, E. Kiss1, T. Krenacs1 1 Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest, Hungary, 2Szent Istvan University, Faculty of Veterinary Science, BudapestFaculty of Veterinary, Hungary Objective: Colorectal cancer is one of the three most common tumor in both men and women. At least 20 % of the patients have distant metastasis at the time of diagnosis, and at this stage the survival rate is only 12%. Extending the local effect to systemic immunological effect is one of the main focus of cancer treatment. Electric field and the concomitant heat (modulated electrohyperthermia, - mEHT) can synergistically provoke cell death in tumor tissue. Here we studied the molecular mechanism required for immunological effect using the herbal extract Marsdenia tenacissima, in combination with mEHT treatment in vivo in colorectal cancer model. Methods: C26 murine colorectal carcinoma cells allografted in both femoral regions of BalbC mice. The treatment groups were (1) single shot mEHT treatment for 30 min on the right side tumor, (2) intraperitoneal injection of 7.5 ml/kg Marsdenia tenacissima extract (MTE) (3) combination of MTE before mEHT and (4) sham control group. In the treatment groups sampling was carried out 12, 24, 48 and 72 h post-treatment. Histomorphologic, immunohistochemical (cleaved caspase-3, cytochrome c, Hsp70, HMGB1, CD3, S100) and TUNEL assay results were analyzed in tissue micro array (TMA) and whole cross sections using digital slides. Results: Modulated electrohyperthermia treatment induced significant cell death in the right tumor center while Marsdenia tenacissima in combination with mEHT caused significant cell death within tumors both in the treated and untreated legs of the same animals. Activated caspase-3 and TUNEL positive cells were detected in mEHT treated tumors and in both tumor tissues of combination treatment 24 h post-treatment with the mitochondrial release of cytochrome c. The nuclear release of HMGB1 protein was also revealed in the combination treatment group. Elevated number of S100 positive cells were found 48 h post-treatment and CD3 positive cells were detected 72 h post-treatment in the mEHT treatment group right tumors and in the combinational group both sides. Conclusion: In our in vivo colorectal model, mEHT in combination with Marsdenia tenacissima extract caused a dominantly caspase dependent programmed cell death in both tumors of the same animals. In line with this elevated number of dendritic cells were observed together with appearance of T cells, indicating that this combination treatment can also induce systemic effects leading to distant tumor destruction in C26 allograft model. Conflict of interest: No conflict of interest.

57 Synergy of combined immune checkpoint modulation and CD30/CD16A-TandAb-induced NK-cell-mediated target cell lysis enhances tumor eradication in vivo X. Zhao1, N. Rajasekaran2, U. Reusch3, J.P. Marschner3, M. Treder3, H. Kohrt2 1 Guiyang Medical University, Department of Immunology, Guiyang, China, 2Stanford University, Center for Clinical Sciences Research, Stanford, USA, 3Affimed GmbH, R&D, Heidelberg, Germany Background: AFM13 is a CD30/CD16A-bispecific tetravalent antibody construct (TandAb) that recruits natural killer (NK) cells to induce rapid target cell lysis of CD30+ tumors. While AFM13 has shown promising clinical activity in patients with CD30+ Hodgkin lymphoma (HL) and is

currently being investigated in phase II, modulation of immune checkpoints has emerged as an effective therapeutic strategy in a variety of cancers, including HL. Here we investigated potential synergy of AFM13 treatment with checkpoint modulation in a patient-derived xenograft model of CD30+ tumors. Materials and methods: AFM13-induced CD30+ target cell lysis was tested in vitro with or without anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies using human PBMCs or enriched NK-cells as effector cells. To assess possible synergy of AFM13 treatment and checkpoint modulation on tumor eradication in vivo, patient-derived CD30+ HL tumor fragments were grafted into immune-deficient mice followed by reconstitution with autologous patient-derived PBMC. Mice were then treated with either AFM13 or anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies alone, or with AFM13 in combination with these immuno-modulatory antibodies before tumor size, tumor-infiltrating lymphocytes and intra-tumoral cytokines were evaluated on day 58. Results and discussion: AFM13 induced robust effector-to-target cell-dependent lysis of up to 40% of CD30+ lymphoma cells in vitro at suboptimal concentrations as a single agent or when combined with antiCTLA-4. Combination with anti-PD-1, anti-CD137, or both substantially enhanced specific lysis to about 50–70% and 80%, respectively. Strikingly, AFM13 treatment resulted in significant reduction of tumor size in vivo when combined with PD-1 blockade or CD137 co-stimulation. The observed tumor shrinkage in the combination regimens exceeded that seen with either agent alone and was correlated with increased numbers of infiltrating NKand T-cells and production of intra-tumoral cytokines. Conclusion: These findings suggest combination of AFM13 with anti-PD-1 immune checkpoint blockade or anti-CD137 co-stimulation may result in significantly improved clinical antitumor responses in HL patients and warrant further investigation of these combinations in clinical trials. The presented data also imply checkpoint modulation may be a universal strategy to significantly enhance therapeutic activity through a direct effect on NK-cell cytotoxicity and induction of immune cell cross-talk. Conflict of interest: Corporate-sponsored Research: This work was financially supported by Affimed GmbH. Other Substantive Relationships: Uwe Reusch, Jens-Peter Marschner and Martin Treder are employees of Affimed GmbH.

58 Custom made fibroblast-specific and antibiotic-free interleukin-12 plasmid for eletrotransfer mediated cancer immunotherapy U. Kamensek1, S. Kos1, N. Tesic2, G. Sersa1, M. Cemazar1 1 Researcher, Department of Experimental Oncology- Institute of Oncology Ljubljana, Ljubljana, Slovenia, 2Researcher, University of PrimorskaFaculty of Health Sciences-, Ljubljana, Slovenia Introduction: Gene electrotransfer (GET) of interleukin 12 (IL-12) encoding plasmids has already entered the clinical evaluation in cancer patients. However, to implement this approach in a broader clinical practice, it must be combined with other standard cancer therapies or vaccination regimes, and it must be approved by regulatory agencies. In the present study our aim was to prepare an optimized IL-12 plasmid for eletrotransfer mediated cancer immunotherapy. In the light of previous studies, showing that for optimal efficacy IL-12 should be released in tumor localized, paracrine manner, we replaced the constitutive ubiquitous promoter with a week endogenous promoter of the collagen 2 gene (COL). Next, to comply with raising regulatory demands for the clinically used plasmids, the antibiotic resistance gene (AB) was removed from the plasmid’s backbone. Materials and methods: The plasmid encoding IL-12 gene under the transcriptional control of the COL promoter was prepared using standard molecular cloning methods and Operator repressor technology (ORTtm, Cobra:bio) was used to prepare the AB free version of the plasmid. The quality and production yields of the prepared plasmids were determined spectrophotometrically and by restriction analysis and sequencing. The



3rd Immunotherapy of Cancer Conference / European Journal of Cancer 55S1 (2016) S1–S30

cytotoxicity of the plasmids was tested by Presto blue cell viability assay, and ELISA was used to determine the expression of IL-12 after GET in mouse endothelial cells (SVEC4-10) and mouse and human fibroblasts (L929, Wi-38) and mouse skin (Balb/c mice). Results and discussion: The plasmid with COL promoter was successfully prepared. Using GET to deliver the plasmid DNA, we showed that COL promoter can drive fibroblast specific expression of IL-12 gene in vitro, and supports localized paracrine secretion of the IL-12 in vivo: in vitro the IL-12 protein levels were statistically higher in the fibroblast cell lines compared to endothelial cell line, and in vivo the levels were increased in the skin, but not in the blood. Next, the expression cassette with IL-12 and COL promoter was cloned in a plasmid lacking the AB. The resulting AB-free plasmid yielded high concentration of pure plasmid DNA and was not cytotoxic; furthermore it demonstrated conserved expression levels and specificity. Conclusion: We prepared an expression plasmid encoding IL-12 under a fibroblast specific promoter and without AB resistance. We proved its feasibility by determining its production yields, and cytotoxicity, tissue specificity, and expression profile after GET in different cell lines in vitro and mouse skin in vivo. The COL promoter and the localized paracrine secretion of IL-12 it supports, makes our plasmid especially usable as adjuvant to anticancer vaccination. The lack of the AB gene makes it applicable for broader clinical use and the initiation of clinical studies. Conflict of interest: No conflict of interest.

59 Use of monoclonal antibodies against a membrane tetraspanin for metastasis control of colorectal cancer R. Grenfell1, N. Almeida1, W. Jeremias1, D. Taboada1, V. Silva-Moraes1, R. Davis2 1 Fundação Oswaldo Cruz, CPqRR, Belo Horizonte, Brazil, 2University of Georgia, Life Sciences, Athens, USA Introduction: The tetraspanin used here is a transmembrane glyco­ protein with 4 hydrophobic domains. When activated on the cellular surface by integrin ligation, it bias to a signal transduction that regulates the cellular development, including activation, grow capacity and motility. The gene responsible for its codification is expressed in different carcinomas, as colorectal cancer, promoting a very invasive metastasis. Within this work, we aim to design a new prototype of immunochemotherapy for colorectal cancer patients. Materials and methods: The sequence encoding the human gene was optimized for mammalian codon usage by utilizing the codons predicted to occur frequently in highly expressed genes of mammals. After the human gene was constructed, it was cloned into the vector and subsequently constructed in HEK293S tetracycline inducible stable cell lines. PCR program for gene synthesis consisted of 30 cycles. In vitro, in vivo and ex vivo procedures are being performed. SW480 and HT29 rectal carcinoma cells were used for inducting cancer in nude mice. Monoclonal antibodies (mAbs) are being produced against the recombinant protein and its specificity will be confirmed by western blotting. Treatments include a PBS control group, a group for each produced mAb anti-tspan and a group for each mAb + VacSIM as an immune adjuvant and vehicle. Results and discussion: The tumor was biolocalized by fluorescence after 24 and 48 h post-induction. Tumor cell was analyzed in vitro and its growth profile was determined. This procedure will follow profile analysis after each treatment in an ex vivo methodology. Recombinant glycoprotein was produced and was injected in BALB/c mice to mAbs production. ELISA showed a successful murine immunization and clone selection is now being performed. Conclusion: We hope to address immunochemotherapy to colorectal invasive cancer in murine model at this time and, in a near future, as a translational research working with samples deposited in a Biobank we have full access.

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Conflict of interest: No conflict of interest.

60 A novel LSD1/HDAC inhibitor 4SC-202 inhibits immunosuppressive MDSC and enhances immunogenicity of tumor cells by up-regulation of TAA expression S. Hamm1, K. Kronthaler2, U. Parnitzke1, T. Prenzel2, H. Kohlhof2, D. Vitt2, R. Baumgartner2 1 4SC Discovery GmbH, Biology, Martinsried/Munich, Germany, 24SC AG, Translational Pharmacology, Martinsried/Munich, Germany Background: Combination of epigenetic agents like DNA methyl­ transferase inhibitor 5-azacytidine or histone deacetylase (HDAC) inhibitors with different cancer immunotherapy approaches were shown to be successful, suggesting these agents themselves engage and mediate an anti-tumoral immune response during cancer therapy. Epigenetic agents modulate gene expression in tumor cells and are able to induce or increase expression of usually silenced genes like tumor associated antigens and stress-induced NKG2D ligands. Here, we showed that the novel combined LSD1 and HDAC inhibitor 4SC-202 displays promising immunomodulating effects potentially contributing to its anti-tumoral activity. Methods: Effects of 4SC-202 on expression of tumor associated antigens (TAAs) were analyzed in K562 (CML), NCI-H69 (SCLC), and in DAOY cells (medulloblastoma). Myeloid derived suppressor cells (MDSCs) were generated by co-culturing human PBMCs with Caki-2 cells for 7 days, and subsequent isolation of CD33+ myeloid cells. The isolated CD33+-population was CD13+/CD14low with ~70–80% of cells being MHC class II negative MDSC. To determine the effect of 4SC-202 on immunosuppressive activity of MDSCs the compound was added for the first 5 days of the co-culture. Results and discussion: At clinically relevant concentrations 4SC-202 induced significant upregulation of TAA in tumor cells which was sustained for at least 2 days after wash out of the compound. Furthermore, in contrast to reference HDAC inhibitors SAHA and entinostat, 4SC-202 did not reduce the viability of stimulated T cells. This indicates that while increased TAA expression may enhance tumor cell recognition by anti-tumoral T cells, T cells were not negatively affected by the presence of the compound. Additionally, 4SC-202 strongly affected immunosuppressive characteristics of MDSCs, since treatment of MDSC culture with this compound enhanced the number of myeloid cells with high MHC class II (HLA-DR) expression from 20% to nearly 90% potentially converting immunosuppressive MDSCs into mature antigen-presenting cells. This effect was additionally emphasized by down-regulation of IDO expression and arginase activity to the basal level of PBMCs. Conclusion: 4SC-202 enhances TAA expression on tumors cells and strongly inhibits immunosuppressive phenotype of myeloid derived suppressor cells suggesting potential synergism with immune checkpoint blocking antibodies (PD1/PDL1 and CTLA-4 inhibitors). Conflict of interest: Other Substantive Relationships: Authors are employees of 4SC group developing 4SC-202 for cancer therapy.

62 Feasibility study of NK cell therapy against cancer T. Shinohara1, J. Masuyama1, K. Tada1, Y. Sato1, S. Uyama1, Y. Takaue1, Y. Heike1 1 St. Luke’s International Hospital, Immunotherapy and Cell Therapy Service, Tokyo, Japan Introduction: Natural killer (NK) cell is a promising tool for cancer immunotherapy because of their potent killing activity against broad range of tumor cells and a capacity to promote anti-tumor immunity. Cellex Co. Ltd. developed a novel method for ex vivo NK cell expansion and has

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applied that against series of cancers from 2004 in New City Osaki Clinic (NCOC). Materials and methods: Peripheral blood mononuclear cells were stimulated with anti-CD3 MAb. and anti-CD52 MAb in NKGM medium (Kohjin Bio) without accessory cells in CO2-permeable bags with autologous plasma and interleukin-2. After 14 days of culture, NK cell (CD3-CD56+) yield was 5.9 × 109 cells (range; 1.6–11.0 × 109 cells, n = 25) from 20 ml peripheral blood of healthy donors, and its median expansion ratio was 859fold (range; 296- to 2700-fold) of the initial NK cells. In vitro cytotoxicity against K562 was 60.3% (median, n = 5) at an E:T ratio of 1:1. A clinical protocol for NK cell therapy was approval of the NCOC ethical committee, and 448 cancer patients were enrolled until May 2011. The studies were designed to estimate the safety and feasibility of NK cell therapy. Results and discussion: Only transient fever (grade 1, 1–2 h after the infusion) was an adverse event we experienced. A survival benefit was evaluated in 33 patients with advanced pancreatic cancer who received at least 4 dose of NK cells combined with/without chemotherapy (gemcitabine and/or S-1). The median overall survival was 16.0 months (range: 4–52) and the 1-year survival rate was 64%. Immunologic monitoring revealed a significant increase in both NK activity and the number of NK cells and NKG2D+ cells in the patients showing clinical benefit. Even though our study showed limited information because that was non-GCP, single-arm study, the result suggested the usefulness of NK cell therapy in cancer treatment. Conclusion: Based on the results of the feasibility studies, We decided to start NK cell therapy registration study. We have already developed a new NK cell expansion kit and a cell process monitoring software (CP-1) to develop well controlled cell processing and NK cell products. We are planning multicenter cell therapy trials in Asia area. Conflict of interest: No conflict of interest.

Poster Session: Anti-Cancer Vaccines

63 Reflating Coley: Active metronomic fever therapy using PRRL and approved drugs U. Hobohm1 1 THM University of Applied Sciences Giessen, Bioinformatics, 35390 Giessen, Germany More than 100 years ago William Coley and contemporaries achieved many cancer remissions by injecting bacterial extracts over weeks and month. Their successes remain a conundrum to this day. In 2008 we proposed that PRRL-substances might be the main molecular triggers for the immune stimulation against cancer cells induced by “Coley‘s toxins“ more than 100 years ago [1]. In 2013, we found strong support for this hypothesis: We could cure cancer mice by metronomically applying a mixture of PRRL (10 times over 3 weeks [2]). We are in contact with private clinics physicians who still apply fever therapy using mistletoe or bacterial extracts. Misteltoe usually is too weak an immune stimulator against cancer cells, while bacterial extracts face increasing scrutiny by regulatory authorities. PRRL could be an alternative, as our mouse experiments have shown. Because purification of PRRL in GMP quality requires substantial investments, we have suggested to use approved drugs who are known to induce fever and contain bacterial extracts. Here we present first data indicating that approved drugs might be substitutes for Coley’s toxins. References: [1] Crit Rev Immunol 2008;28(2):95–107 [2] Cancer Immunol Immunother 2013;62:1283–92 Conflict of interest: No conflict of interest.

64 Activation of the cytosolic RNA receptor RIG-I in tumor and immune cells triggers efficient anti-tumor immunity and synergizes with checkpoint blockade S. Heidegger1, W. Alexander1, D. Kreppel1, M. Bscheider1,2, S. Bek1, J. Fischer1, M. Schmickl1, J. Ruland3, C. Peschel1, T. Haas1, H. Poeck1,4 1 Klinikum rechts der Isar- TU München, 3. Medizinische Klinik, München, Germany, 2Stanford University School of Medicine, Department of Pathology, Stanford, USA, 3Klinikum rechts der Isar- TU München, Institut für Klinische Chemie und Pathobiochemie, München, Germany, 4 Memorial Sloan-Kettering Cancer Center, Departments of Immunology and Medicine- and Cell Biology, New York, USA Introduction: Targeting inhibitory T-cell receptors such as CTLA-4 has shown great promise in cancer therapy but the therapeutic success is limited to a minority of patients. Checkpoint inhibitors seem to synergize with treatment strategies that induce the production of type I IFNs. In contrast to other cytosolic nucleic acid receptors involved in type I IFN release (i.e. cGAS-STING pathway), selective activation of the RLH family member RIG‑I has been shown to induce an immunogenic variant of cancer cell death that presumably activates innate and adaptive immune responses. However, the molecular mechanisms in the tumor microenvironment that eventually lead to RIG-I-induced tumor growth inhibition remain unknown. Materials and methods: Mice bearing bilateral B16 melanoma tumors were treated by unilateral intratumoral injections with the specific RIG-I ligand 3pRNA in combination with anti-CTLA-4. Tumor growth and immune cell function were monitored. Using CRISPR/Cas9 technology to generate RIG-I-deficient B16 melanoma cells and available genetically deficient mouse models, we addressed the importance of tumor-intrinsic and host immune cell RIG-I signaling for efficient anti-tumor immunity in combination with anti-CTLA-4. Results and discussion: We show that therapeutic activation of RIG-I synergizes with CTLA-4 checkpoint blockade. Selective RIG-I ligation in the tumor microenvironment induced strong in situ vaccination with expansion of tumor-specific CD8+ T cells that resulted in rejection of local and distant non-immunogenic tumors that are resistant to anti-CTLA-4 mono-therapy. Fully efficient anti-tumor immunity was dependent on RIG-I activation in both malignant and host immune cells in the tumor microenvironment. Furthermore, protein vaccination together with RIG-I ligation and anti-CTLA-4 induced expansion of antigen-specific CD8+ T cells that translated into potent anti-tumor immunity. Cross-priming of cytotoxic T cells as well as anti-tumor immunity were critically dependent on the adapter protein MAVS and host type I IFN signaling and were mediated by dendritic cells. Conclusion: We here show that RIG-I activation either in combination with a cancer vaccine or selectively engaged in the tumor microenvironment strongly synergizes with CTLA-4 blockade. Fully efficient anti-tumor immunity is dependent on RIG-I signaling in both malignant and host immune cells in the tumor microenvironment. Thus, our data may serve as the basis for new combinatorial approaches in the immunotherapy of human cancers. Conflict of interest: No conflict of interest.

65 Next-generation dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I/II trial K. Deiser1,2, F. Lichtenegger1,2, F. Schnorfeil1,2, T. Köhnke2, T. Altmann2, V. Bücklein2, A. Moosmann3, M. Brüggemann4, M.H.M. Heemskerk5, B. Wagner6, W. Hiddemann2, I. Bigalke7, G. Kvalheim7, M. Subklewe1,2 1 Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany, 2Klinikum der Universität München, Department of Internal Medicine III, Munich, Germany, 3Helmholtz Zentrum München, Clinical Cooperation Group Immunooncology,