Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor gene loss. Given the type 1 interferon regulatory function of BRCA1 and PTENgenes and their associated contrasting T-cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, respectively, in this study we investigated the potential of stimulator of interferon genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency. METHODS: Expression of PTEN protein was evaluated in tissue microarrays generated using pretreatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8-Trp53 -/-; Pten -/- and ID8-Trp53 -/-; Brca1 -/-, were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy. RESULTS: Patient tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intraepithelial CD68+ macrophage infiltration as compared with intact PTEN expression. In vivo studies demonstrated that Pten-deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+MDSCs in the ascites, and reduced effector CD8+ cytotoxic T-cell function compared with Brca1-deficient cells; further, tumors from mice injected with Pten-deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten-deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten-deficient ascites to M1-like phenotype and rescued CD8+ cytotoxic T-cell activation. CONCLUSIONS: This study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN-deficient HGSC.

Inhibiting DNA methylation and RNA editing upregulates immunogenic RNA to transform the tumor microenvironment and prolong survival in ovarian cancer.

BACKGROUND: Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS: Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days. RESULTS: We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-ß compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION: In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells.

BACKGROUND: Dendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs. METHODS: We first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation. RESULTS: We show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8+ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration. CONCLUSIONS: Our data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer.

TGFß receptor inhibition unleashes interferon-ß production by tumor-associated macrophages and enhances radiotherapy efficacy.

BACKGROUND: Transforming growth factor-beta (TGFß) can limit the efficacy of cancer treatments, including radiotherapy (RT), by inducing an immunosuppressive tumor environment. The association of TGFß with impaired T cell infiltration and antitumor immunity is known, but the mechanisms by which TGFß participates in immune cell exclusion and limits the efficacy of antitumor therapies warrant further investigations. METHODS: We used the clinically relevant TGFß receptor 2 (TGFßR2)-neutralizing antibody MT1 and the small molecule TGFßR1 inhibitor LY3200882 and evaluated their efficacy in combination with RT against murine orthotopic models of head and neck and lung cancer. RESULTS: We demonstrated that TGFß pathway inhibition strongly increased the efficacy of RT. TGFßR2 antibody upregulated interferon beta expression in tumor-associated macrophages within the irradiated tumors and favored T cell infiltration at the periphery and within the core of the tumor lesions. We highlighted that both the antitumor efficacy and the increased lymphocyte infiltration observed with the combination of MT1 and RT were dependent on type I interferon signaling. CONCLUSIONS: These data shed new light on the role of TGFß in limiting the efficacy of RT, identifying a novel mechanism involving the inhibition of macrophage-derived type I interferon production, and fostering the use of TGFßR inhibition in combination with RT in therapeutic strategies for the management of head and neck and lung cancer.

Immunostimulatory effects of radioimmunotherapy.

Radiation therapy (RT) is known for its ability to kill cancer cells in an immunogenic manner. Recent preclinical data demonstrate that targeted alpha-particle therapy shares with RT the capacity to elicit immunostimulatory effects, standing out as a promising strategy to circumvent immune checkpoint inhibitor resistance in immunologically 'cold' tumors.

Elucidating mechanisms of antitumor immunity mediated by live oncolytic vaccinia and heat-inactivated vaccinia.

BACKGROUND: Viral-based immunotherapy can overcome resistance to immune checkpoint blockade (ICB) and fill the unmet needs of many patients with cancer. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study was to compare the antitumor effects of live oncolytic vaccinia viruses versus the inactivated versions and elucidate their underlying immunological mechanisms. METHODS: We engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase locus of a mutant vaccinia E3L∆83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM versus heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model. We also generated vvDD, a well-studied oncolytic vaccinia virus, and compared the antitumor effects of live vvDD vs heat-inactivated vvDD (heat-ivvDD) in a murine A20 B-cell lymphoma bilateral tumor implantation model. RESULTS: Heat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induced type I interferon and proinflammatory cytokines and chemokines, whereas live OV-GM did not. IT live OV-GM was less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. When combined with systemic delivery of ICB, IT heat-iOV-GM was more effective in eradicating tumors, compared with live OV-GM. IT heat-ivvDD was also more effective in treating murine A20 B-cell lymphoma, compared with live vvDD. CONCLUSIONS: Tumor lysis induced by the replication of oncolytic vaccinia virus has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM, with the latter being more potent than live OV-GM in inducing innate and adaptive immunity in both locally injected and distant, non-injected tumors. We propose that evaluations of both innate and adaptive immunity, induced by IT oncolytic viral immunotherapy at injected and non-injected tumors, should be included as potential biomarkers for host responses to viral therapy.

Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors.

BACKGROUND: Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms. METHODS: In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/ß receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response. RESULTS: In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/ß receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen. CONCLUSION: In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.

Oncolytic parainfluenza virus combines with NK cells to mediate killing of infected and non-infected lung cancer cells within 3D spheroids: role of type I and type III interferon signaling.

BACKGROUND: There is intense interest in developing novel oncolytic viruses, which can be used in cancer therapies along with immune cells such as natural killer (NK) cells. We have previously developed a particle-based method for in vitro expansion of highly cytotoxic human NK cells (PM21-NK cells). Here, we have tested the hypothesis that oncolytic parainfluenza virus 5 (P/V virus) can combine with PM21-NK cells for targeted killing of lung cancer cells. METHODS: PM21-NK cells were assayed for killing of P/V virus-infected A549, H1299 and Calu-1 lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cultures using flow cytometry, luminescence and kinetic imaging-based methods. Blocking antibodies were used to evaluate NK cell activating receptors involved in PM21-NK cell killing of infected target cells. Media transfer experiments tested soluble factors that increase PM21-NK cell killing of both P/V virus-infected and uninfected tumor cells. RESULTS: In 2D cultures, PM21-NK cells efficiently killed P/V virus-infected cancer cells compared with non-infected cells, through involvement of the viral glycoprotein and NK cell receptors NKp30, NKp46 and NKG2D. In 3D spheroid cultures, P/V virus infection was restricted to the outer layer of the spheroid. However, PM21-NK cells were able to more efficiently kill both the outer layer of infected cells in the spheroid and progressing further to kill the uninfected interior cells. Media transfer experiments demonstrated that P/V virus infection produced both type I and type III interferons, which decreased cell growth, which contributed to a reduction in the overall number of uninfected tumor cells in conjunction with PM21-NK cells. Across five cancer cell lines, the contribution of P/V virus infection on PM21-NK cell killing of target cells correlated with interferon induction. CONCLUSION: Our data support the potential of combining oncolytic parainfluenza virus with PM21-NK cell adoptive therapy against lung cancer.

STING activation normalizes the intraperitoneal vascular-immune microenvironment and suppresses peritoneal carcinomatosis of colon cancer.

BACKGROUND: Peritoneal carcinomatosis is a fatal clinical presentation of colon cancer, characterized by unresponsiveness to conventional anticancer therapies, including immune checkpoint inhibitors. Here, we elucidated the immune-evasion mechanisms during the peritoneal carcinomatosis of colon cancer and developed a novel immunotherapy by activating the stimulator of interferon genes (STING) pathway. METHODS: We generated a syngeneic peritoneal carcinomatosis model of colon cancer. Mice were intraperitoneally treated with either STING agonist (MIW815, also known as ADU-S100) or PD-1 blockade or both. The tumor microenvironment was comprehensively analyzed using multiplexed immunofluorescence imaging, flow cytometry, and NanoString immune profiling. RESULTS: Intraperitoneal colon cancer cells induce a massive influx of immunosuppressive M2-like macrophages, upregulate immune checkpoints, and impair effector T cell functions during peritoneal dissemination; these collectively create a highly angiogenic and immunosuppressive milieu that is resistant to anti-PD-1 monotherapy. Intraperitoneal administration of a STING agonist suppressed aberrant angiogenesis, increased pericyte coverage, and normalized tumor vessels, thereby facilitating the infiltration of activated CD8+ T cells into peritoneal tumor nodules. Moreover, STING activation reprogramed tumor-associated macrophages toward the M1 phenotype. STING activation converted immunologically cold peritoneal tumors into T-cell-inflamed tumors in a type-I interferon-dependent manner. Lastly, the STING agonist synergistically cooperated with PD-1 and/or COX2 blockade to further suppress the peritoneal dissemination of colon cancer, resulting in complete eradication of tumor and ascites, and inducing durable antitumor immunity. CONCLUSIONS: STING activation can normalize the peritoneal vascular and immune microenvironment, providing a rationale for a novel combination therapeutic strategy for peritoneal carcinomatosis in colon cancer.

Transcriptional analysis of multiple ovarian cancer cohorts reveals prognostic and immunomodulatory consequences of ERV expression.

BACKGROUND: Endogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC. METHODS: ERV expression was assessed using EOC transcriptional data from The Cancer Genome Atlas (TCGA) and from an independent cohort (Hammersmith Hospital, HH), as well as from untreated or DNMTi-treated EOC cell lines. Least absolute shrinkage and selection operator (LASSO) logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells. RESULTS: ERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+T cells (r=0.46, p=0.0001). In the TCGA dataset, a higher ERV score was found in BRCA1/2 mutant tumors, compared to wild type (p=0.015), while a lower ERV score was found in CCNE1 amplified tumors, compared to wild type (p=0.019). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of an ERV expression threshold by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells. CONCLUSIONS: These findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect antitumor immunity.

Mitochondrial Lon-induced mtDNA leakage contributes to PD-L1-mediated immunoescape via STING-IFN signaling and extracellular vesicles.

BACKGROUND: Mitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production of mitochondrial reactive oxygen species (ROS). However, there is little information in detail on how mitochondrial Lon regulates ROS-dependent cancer immunoescape through mtDNA metabolism in the tumor microenvironment (TME). METHODS: We explored the understanding of the intricate interplay between mitochondria and the innate immune response in the inflammatory TME. RESULTS: We found that oxidized mtDNA is released into the cytosol when Lon is overexpressed and then it induces interferon (IFN) signaling via cGAS-STING-TBK1, which upregulates PD-L1 and IDO-1 expression to inhibit T-cell activation. Unexpectedly, upregulation of Lon also induces the secretion of extracellular vehicles (EVs), which carry mtDNA and PD-L1. Lon-induced EVs further induce the production of IFN and IL-6 from macrophages, which attenuates T-cell immunity in the TME. CONCLUSIONS: The levels of mtDNA and PD-L1 in EVs in patients with oral cancer function as a potential diagnostic biomarker for anti-PD-L1 immunotherapy. Our studies provide an insight into the immunosuppression on mitochondrial stress and suggest a therapeutic synergy between anti-inflammation therapy and immunotherapy in cancer.

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse.

BACKGROUND: Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. METHODS: We designed a mouse model of tumor relapse in which mice transplanted with E7+ TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. RESULTS: We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. CONCLUSIONS: Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.

Preconditioning of the tumor microenvironment with oncolytic reovirus converts CD3-bispecific antibody treatment into effective immunotherapy.

BACKGROUND: T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. METHODS: The mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. RESULTS: Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. CONCLUSIONS: Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

BCG vaccine and COVID-19: implications for infection prophylaxis and cancer immunotherapy.

The COVID-19 pandemic has killed over 400 000 people globally. Ecological evidence indicates that countries with national universal BCG vaccination programs for tuberculosis (TB) prevention have a much lower incidence of severe COVID-19 and mortality compared with those that do not have such programs. BCG is a century old vaccine used for TB prevention via infant/childhood vaccination in lowto middle-income countries with high infection prevalence rate and is known to reduce all-cause neonatal mortality. BCG remains the standard immunotherapy treatment for patients with high-risk non-muscle invasive bladder cancer globally for more than 44 years. Several trials are, therefore, investigating BCG as a prophylactic against COVID-19 in healthcare workers and the elderly. In this commentary, we discuss the potential mechanisms that may underlie BCG associated heterologous protection with a focus on tertiary lymphoid structure (TLS) organogenesis. Given the significance of TLSs in mucosal immunity, their association with positive prognosis and response to immune checkpoint blockade with a critical role of Type I interferon (IFN-1) in inducing these, we also discuss potentiating TLS formation as a promising approach to enhance anti-tumor immunity. We propose that lessons learned from BCG immunotherapy success could be applied to not only augment such microbe-based therapeutics but also lead to similar adjunctive IFN-1 activating approaches to improve response to immune checkpoint blockade therapy in cancer.

[Evaluation of the effectiveness of ARVI treatment regimen including etiotropic (enisamium iodide) and symptomatic treatment].

AIM: To assess the effectiveness of the use of the antiviral drug enisamium iodide in the complex treatment of acute respiratory viral infections (ARVI) caused by various pathogens in routine clinical practice. MATERIALS AND METHODS: А prospective randomized study included 134 patients who were treated in the epidemic season of influenza and ARVI in 20182019. All patients were examined for the presence of influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, rhinoviruses, adenoviruses in nasopharyngeal swabs by PCR. Patients of the main group received enisamium iodide along with symptomatic therapy, the control group received only symptomatic therapy. The primary parameter of the effectiveness of therapy was evaluated on the scale of the general severity of the manifestations of ARVI (Total Symptom Score TSS) from the 2nd to the 4th day and by the secondary criteria of effectiveness: assessment of the duration of ARVI, the severity of fever, the proportion of patients with normal body temperature, the duration of the main clinical symptoms of acute respiratory viral infections, the proportion of patients in whom complications requiring antibiotics were noted, the dynamics of interferon status on the 6th day. To conduct a statistical analysis, depending on the efficiency parameter, the ANCOVA method with a fixed group factor and an initial score on the TSS severity scale was used as covariates, a criterion for comparing quantitative indicators in two independent groups. RESULTS: According to the results of the analysis of the primary efficacy parameter, the median (interquartile range) of the average score on the scale of the general severity of ARVI manifestations in the main group was 4.33 (3.675.83), in the comparison group 6.00 (4.677.25; p0.001). The duration of systemic and local manifestations of acute respiratory viral infections was statistically significantly less in the main group (p=0.002 and p=0.019, respectively). Prescription of additional therapy was required in 2 (2.9%) patients of the main group (patients taking enisamium iodide), compared with 8 (11.9%) patients in the control group. Serum levels of interferon  and interferon  on the last day of treatment were statistically significantly higher in patients of the main group compared with the control group (p0.001). Treatment (excellent) was evaluated by 42 (62.7%) patients, while in the control group only 17 (25.8%) patients gave similar ratings. Both patients (p0.001) and doctors (p0.002) rated therapy tolerance better in the study group. CONCLUSION: The results confirmed the safety and effectiveness of enisamium iodide as a treatment for ARVI and influenza. The antiviral, interferonogenic and anti-inflammatory properties of the drug are involved in the formation of an antiviral response and reduce the risk of complications, which makes it possible to reduce the number of symptomatic agents used.

Dual oxidase 1 limits the IFNγ-associated antitumor effect of macrophages.

BACKGROUND: Macrophages play pivotal roles in tumor progression and the response to anticancer therapies, including radiotherapy (RT). Dual oxidase (DUOX) 1 is a transmembrane enzyme that plays a critical role in oxidant generation. METHODS: Since we found DUOX1 expression in macrophages from human lung samples exposed to ionizing radiation, we aimed to assess the involvement of DUOX1 in macrophage activation and the role of these macrophages in tumor development. RESULTS: Using Duox1-/- mice, we demonstrated that the lack of DUOX1 in proinflammatory macrophages improved the antitumor effect of these cells. Furthermore, intratumoral injection of Duox1-/- proinflammatory macrophages significantly enhanced the antitumor effect of RT. Mechanistically, DUOX1 deficiency increased the production of proinflammatory cytokines (IFNγ, CXCL9, CCL3 and TNFα) by activated macrophages in vitro and the expression of major histocompatibility complex class II in the membranes of macrophages. We also demonstrated that DUOX1 was involved in the phagocytotic function of macrophages in vitro and in vivo. The antitumor effect of Duox1-/- macrophages was associated with a significant increase in IFNγ production by both lymphoid and myeloid immune cells. CONCLUSIONS: Our data indicate that DUOX1 is a new target for macrophage reprogramming and suggest that DUOX1 inhibition in macrophages combined with RT is a new therapeutic strategy for the management of cancers.

Tilorone: a Broad-Spectrum Antiviral Invented in the USA and Commercialized in Russia and beyond.

For the last 50 years we have known of a broad-spectrum agent tilorone dihydrochloride (Tilorone). This is a small-molecule orally bioavailable drug that was originally discovered in the USA and is currently used clinically as an antiviral in Russia and the Ukraine. Over the years there have been numerous clinical and non-clinical reports of its broad spectrum of antiviral activity. More recently we have identified additional promising antiviral activities against Middle East Respiratory Syndrome, Chikungunya, Ebola and Marburg which highlights that this old drug may have other uses against new viruses. This may in turn inform the types of drugs that we need for virus outbreaks such as for the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tilorone has been long neglected by the west in many respects but it deserves further reassessment in light of current and future needs for broad-spectrum antivirals.

[Development of drugs on the basis of high-polymeric double-stranded RNA for antiviral and antitumor therapy]. / Sozdanie lekarstvennykh preparatov na osnove vysokopolimernykh dvuspiral'nykh RNK dlia protivovirusnoi i protivoopukholevoi terapii.

The review summarizes literature data on the development of drugs based on natural and synthetic high-polymeric double-stranded RNA, and their antiviral, immunoadjuvant and antitumor properties. Special attention is paid to cell receptors responding to exogenous dsRNA, the paths of dsRNA-dependent antiviral reaction, ability of dsRNA to inhibit growth and induce apoptosis ofmalignant cells. It has been shown that enhancing the innate immune response with dsRNA can be an effective component in improving methods for treating and preventing infectious and cancer diseases. The further use of dsRNA for the correction of pathological processes of different origin is discussed.

Development of Drugs Based on High-Polymeric Double-Stranded RNA for Antiviral and Antitumor Therapy.

Abstract-The review summarizes literature data on the development of drugs based on natural and synthetic high-polymeric double-stranded RNA (dsRNA), their antiviral, immunoadjuvant, and antitumor properties. Special attention is paid to cell receptors responding to exogenous dsRNA, pathways of dsRNA-dependent antiviral reaction, ability of dsRNA to inhibit growth and induce apoptosis of malignant cells. It has been shown that enhancing the innate immune response with dsRNA can be an effective component in improving methods for treating and preventing infectious and cancer diseases. The further use of dsRNA for the correction of pathological processes of different origin is discussed.

THEORETICAL AND APPLIED ASPECTS OF THE INTERFERON SYSTEM: TO THE 60TH ANNIVERSARY OF THE DISCOVERY OF INTERFERONS.

The review contains a brief analysis of the 60-year history of the discovery, study and medical application of interferons, a new group of remarkable proteins that have found wide medical application in the therapy of virological, oncological, neurological, ophthalmic and other pathologies. Modern data on the classification of interferons and the mechanisms of their action are given. Particular attention is paid to the clinical use of medications of interferon and its inducers.