Background Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 enhances antitumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. Methods Here we established a vaccination strategy that combines engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4 blockade. We used in vitro transcribed 5′-triphosphorylated RNA (3pRNA) to therapeutically target the RIG-I pathway. We performed in vitro functional analysis in bone-marrow derived dendritic cells and investigated RIG-I-enhanced vaccines in different murine melanoma models. Findings We found that protein vaccination together with RIG-I ligation via 3pRNA strongly synergizes with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8 + T cells that translates into potent antitumor immunity. RIG-I-induced cross-priming of cytotoxic T cells as well as antitumor immunity were dependent on the host adapter protein MAVS and type I interferon (IFN-I) signaling and were mediated by dendritic cells. Interpretation Overall, our data demonstrate the potency of a novel combinatorial vaccination strategy combining RIG-I-driven immunization with CTLA-4 blockade to prevent and treat experimental melanoma. Fund German Research Foundation (SFB 1335, SFB 1371), EMBO, Else Kröner-Fresenius-Foundation, German Cancer Aid, European Hematology Association, DKMS Foundation for Giving Life, Dres. Carl Maximilian and Carl Manfred Bayer-Foundation.

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Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 has been shown to enhance anti-tumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. Here we established a vaccination protocol that combines selective engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4-blockade. We found that vaccination together with RIG-I ligation strongly synergized with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8+ T cells and potent anti-tumor immunity. Cross-priming of cytotoxic T cells as well as anti-tumor immunity required the adapter protein MAVS and type I interferon (IFN) signaling and were mediated by dendritic cells. In addition, the benefit of the combined immunization with anti-CTLA-4 was reduced by systemic antibiotics pointing to the requisite of an intact commensal microbiota in this context. Together, our findings describe a novel combinatorial strategy that may form the basis for the design of new type I IFN-based regimens that enhance antigen-specific T cell reactivity against cancer. Disclosures No relevant conflicts of interest to declare.

Introduction: Initial studies from Wuhan, China reported patients infected with SARS-CoV-2 have uncontrolled coagulopathy and an increased risk for thrombotic complications, including pulmonary embolism (PE), deep vein thrombosis (DVT), and arterial thrombosis.1 The incidence of thrombosis attributed to coronavirus disease 2019 (COVID-19) ranged from 9.5% in all hospital-admitted patients to 31% in the critically ill.2,3 COVID-19 has had a major impact on the Chicago metropolitan area with over 121,000 confirmed cases as of August 2020, Cook county being the 4th highest affected county after Maricopa, Miami-Dade and Los Angeles counties.4 The primary goal of this study is to describe the rate of thrombotic events in the Chicago metropolitan area, highlighting an ethnically diverse population, and identify new risk factors for thrombosis between three university health systems. Methods: We conducted a retrospective analysis between three university health systems in the Chicago metropolitan area: Loyola University Health System (LUHS): comprised of one tertiary and two community hospitals, Rush University System for Health (RUSH): comprised of one tertiary and two community hospitals, and University of Illinois-Chicago (UIC): a tertiary hospital. All patients had positive SARS-CoV-2 testing and were hospitalized for COVID-19. PE, DVT or arterial thrombosis were confirmed by supportive imaging modalities. Wilcoxon rank sum test were used to test the associations of continuous variables; Chi-square test or Fisher's exact test were used to test the associations of categorical variables. All analyses were performed with SAS 9.4 and two-sided p-value < .05 were deemed statistically significant. Results: Between March and May 2020, 2,180 patients from LUHS, RUSH and UIC were hospitalized for COVID-19 and were included in our analysis. Baseline patient demographics are described in Table 1. Race/ethnicity demographics are as follows: Hispanics (H)/ African Americans (AA) represented 47%/17% of LUHS patients, 32%/42% of RUSH patients, and 36%/51% of UIC patients, respectively (Figure 1). Intensive care admissions were needed in 33% of all patients. Documented total thrombotic events are as follows: LUHS = 5.4% (41 VTE/PE, 10 arterial and 5 with both venous and arterial); RUSH = 9.7% (70 VTE/PE, 7 arterial and 4 with both venous/arterial); UIC = 6% (14 VTE/PE, 4 arterial and 0 with both venous/arterial). Patients that developed a thrombotic event were similar by age, sex, and BMI to those without a thrombotic event. Anticoagulation prophylaxis was given to 82% of pts at LUHS and UIC at time of admission. Collectively, those with thrombotic events (N=156) had higher incidence of intensive care admission, elevated white blood cell (WBC) count and a d-dimer >5X upper limit normal (ULN) at presentation. Furthermore, a higher proportion of pts that had a thrombotic event were diabetic at LUHS and RUSH (Table 2). Mortality in COVID-19 patients was 13-16% and patients that had a thrombotic event had a higher risk of death in the RUSH and UIC cohorts. Conclusions: In a racially diverse, multi-institutional cohort of patients, we demonstrate that 7.2% of COVID-19 patients had a thrombotic event. Consistent risk factors for thrombosis across the different centers included an initial d-dimer levels >5X ULN, elevated initial WBC count, diabetes, and being critically ill. Mortality differences and anticoagulation practices between the institutions as well as race/ethnicity differences regarding thrombosis will be explored in future combined multivariate analyses. Finally, based off these risk factors, identification of patients at most risk for thrombosis is needed to reduce the morbidity and mortality when diagnosed with COVID-19. References -Tang et. al. J Thromb Haemost. 2020;18:844-847. -Klock et. Al. Thrombosis Research 2020;191:145-147. -Al-Samkari H, Laef RS, Dzik WH et. Al. COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection. Blood. 2020;136(4):489-500. -https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/county-map.html; accessed 8/7/20. Disclosures Arain: Astellas: Other: Spouse is employed. Stiff:Macrogenics: Research Funding; Delta-Fly: Research Funding; Unum: Research Funding; Atara: Research Funding; Kite, a Gilead Company: Research Funding; Amgen: Research Funding; Gamida Cell: Research Funding. Saraf:Novartis, Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Boards, Speakers Bureau; Pfizer, Global Blood Therapeutics, Novartis: Research Funding.

Introduction: Targeting inhibitory T-cell receptors such as CTLA-4 has shown great promise in cancer therapy. However, strong inter-individual variation in clinical response to checkpoint inhibitors remains a major challenge. Expression of viral defense genes in human melanomas including the cytosolic RNA receptor RIG-I (DDx58) has recently been associated with clinical benefit to CTLA-4 blockade. Methods: Using CRISPR/Cas9 technology to generate B16 melanoma cells lines that lack nucleic acid receptors or downstream signaling molecules (RIG-I, STING, IRF3/7) together with available genetically deficient mouse models, we address the importance of nucleic acid receptor signaling for the efficacy of anti-CTLA-4 immunotherapy. Results: We provide experimental proof that anti-CTLA-4 immunotherapy indeed relies on tumor cell-intrinsic RIG-I/MAVS but not cGAS/STING signaling. Consistently, therapeutic targeting of RIG-I with the specific ligand 5'‑triphosphate-RNA (3pRNA) potently augments the efficacy of CTLA-4 checkpoint blockade. In situ vaccination by intratumoral 3pRNA injection enhanced cross-presentation of tumor-associated antigens by CD103+ migratory DCs in local draining lymph nodes. Subsequent expansion of tumor-specific CD8+ T cells resulted in control of local and distant tumors, otherwise resistant to anti-CTLA‑4 monotherapy. These processes were critically dependent on tumor cell-intrinsic RIG-I-mediated immunogenic cell death (ICD), but not tumor-derived type I IFN release. In such, the synergistic therapeutic effect of RIG-I activation and anti-CTLA-4 was restricted to tumor models that showed susceptibility to RIG‑I-mediated ICD. Furthermore, systemic antitumor immunity following anti-CTLA-4 +/- 3pRNA treatment required RIG-I signaling in both tumor and non-malignant host cells. Conclusion: Our study identifies a hitherto unrecognized role of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer. Therapeutic targeting of RIG-I using specific agonists strongly augments the efficacy of anti-CTLA-4 blockade and may thus serve as the basis for new combinatorial approaches. Citation Format: Simon Heidegger, Alexander Wintges, Diana Kreppel, Sarah Bek, Michael Bscheider, Martina Schmickl, Marcel R.M. van den Brink, Christian Peschel, Tobias Haas, Hendrik Poeck. Tumor- and host-intrinsic RIG-I signaling promote anticancer immunity by CTLA-4 blockade [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A005.