Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model.

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Introduction: Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. Methods: The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Results and discussion: Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients' primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors.

Mast Cells as a Target-A Comprehensive Review of Recent Therapeutic Approaches.

Mast cells (MCs) are the immune cells distributed throughout nearly all tissues, mainly in the skin, near blood vessels and lymph vessels, nerves, lungs, and the intestines. Although MCs are essential to the healthy immune response, their overactivity and pathological states can lead to numerous health hazards. The side effect of mast cell activity is usually caused by degranulation. It can be triggered by immunological factors, such as immunoglobulins, lymphocytes, or antigen-antibody complexes, and non-immune factors, such as radiation and pathogens. An intensive reaction of mast cells can even lead to anaphylaxis, one of the most life-threatening allergic reactions. What is more, mast cells play a role in the tumor microenvironment by modulating various events of tumor biology, such as cell proliferation and survival, angiogenesis, invasiveness, and metastasis. The mechanisms of the mast cell actions are still poorly understood, making it difficult to develop therapies for their pathological condition. This review focuses on the possible therapies targeting mast cell degranulation, anaphylaxis, and MC-derived tumors.

Oncolytic Adenoviruses Armed with Co-Stimulatory Molecules for Cancer Treatment.

In clinical trials, adenovirus vectors (AdVs) are commonly used platforms for human gene delivery therapy. High genome capacity and flexibility in gene organization make HAdVs suitable for cloning. Recent advancements in molecular techniques have influenced the development of genetically engineered adenovirus vectors showing therapeutic potential. Increased molecular understanding of the benefits and limitations of HAdVs in preclinical research and clinical studies is a crucial point in the engineering of refined oncolytic vectors. This review presents HAdV species (A-G) used in oncotherapy. We describe the adenovirus genome organizations and modifications, the possibilities oncolytic viruses offer, and their current limitations. Ongoing and ended clinical trials based on oncolytic adenoviruses are presented. This review provides a broad overview of the current knowledge of oncolytic therapy. HAdV-based strategies targeting tumors by employing variable immune modifiers or delivering immune stimulatory factors are of great promise in the field of immune oncologyy This approach can change the face of the fight against cancer, supplying the medical tools to defeat tumors more selectively and safely.

Next generation oncolytic viruses expressing PADI1 and TIMP2 exhibit anti-tumor activity against melanoma in nude and humanized mouse models.

Immunotherapy of metastatic melanoma (MM) has vastly improved the longevity of only a minority of patients. To broaden the repertoire of agents against MM, we investigated the effectiveness of locally interrupting tumor blood endothelial cell proliferation and angiogenesis, arginine deprivation, or both on the growth of melanoma by constructing and characterizing the effectiveness of four oncolytic adenoviruses. ONCOS-207 (which expressed tissue inhibitor of metalloprotease type 2 [TIMP2]), ONCOS-209 (which expressed peptidyl arginine deiminase [PADI1]), and ONCOS-210 and ONCOS-212 (which expressed both TIMP2 and PADI1) exhibited oncolytic activity against four melanoma cell lines in vitro. ONCOS-212 treatments significantly inhibited tumor growth in an A2058 tumor model in nude mice compared with vehicle control. The inhibitory effects of the two transgenes of ONCOS-212 on tumor growth appeared to be synergistic. These viruses also significantly inhibited tumor growth in a humanized NOG model of melanoma (A2058 xenograft). All viruses significantly increased the percentage of activated CD8+ T cells in the tumor-infiltrating lymphocytes. The abscopal effect of ONCOS-212 treatments in the A2058 tumor challenge model in hNOG mice supports the hypothesis that the human immune response contributes to the anti-tumor activity of ONCOS-212. These results support the further development of ONCOS-212 for cancer treatment.

Pilot Study of ONCOS-102 and Pembrolizumab: Remodeling of the Tumor Microenvironment and Clinical Outcomes in Anti-PD-1-Resistant Advanced Melanoma.

PURPOSE: Intratumoral oncolytic virotherapy may overcome anti-PD(L)-1 resistance by triggering pro-inflammatory remodeling of the tumor microenvironment. This pilot study investigated ONCOS-102 (oncolytic adenovirus expressing GM-CSF) plus anti-programmed cell death protein 1 (PD)-1 therapy in anti-PD-1-resistant melanoma. PATIENTS AND METHODS: Patients with advanced melanoma progressing after prior PD-1 blockade received intratumoral ONCOS-102 either as priming with 3 doses (3 × 1011 viral particles) during Week 1 [Part 1 (sequential treatment)] or as 4-dose priming and 8 booster doses every 3 weeks [Part 2 (combination treatment)]. From Week 3, all patients received pembrolizumab every 3 weeks (≤8 doses). The primary endpoint was safety. Objective response rate (ORR), progression-free survival, and immunologic activation in repeat biopsies were also investigated. RESULTS: In 21 patients (Part 1, n = 9; Part 2, n = 12) ONCOS-102 plus pembrolizumab was well tolerated: most adverse events (AE) were mild/moderate in severity. Pyrexia (43%), chills (43%), and nausea (28%) were the most common ONCOS-102-related AEs. There were no dose-limiting toxicities. ORR was 35% [response evaluation in solid tumors (RECIST) 1.1, irRECIST]. Reduction in size of ≥1 non-injected lesions observed in 53% patients indicated a systemic effect. In injected tumors, persistent immune-related gene expression and T-cell infiltration were associated with clinical benefit. Viral persistence and efficacy in injected and non-injected lesions without additional toxicity supported Part 2 dosing regimen in future studies. CONCLUSIONS: ONCOS-102 plus pembrolizumab was well tolerated and led to objective responses in patients with anti-PD-1-resistant advanced melanoma. ONCOS-102 promoted T-cell infiltration, particularly cytotoxic CD8+ T cells, which persisted at Week 9, driving clinical benefit. Further investigation of ONCOS-102 plus PD-1 blockade is warranted. See related commentary by Levi and Boland, p. 3.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

From Immunosuppression to Immunomodulation - Turning Cold Tumours into Hot.

Cancer cells employ various mechanisms to evade and suppress anti-cancer immune responses generating a "cold" immunosuppressive tumour microenvironment. Oncolytic viruses are a promising tool to convert tumour immunosuppression to immunomodulation and improve the efficacy of cancer treatment. Emerging preclinical and clinical findings confirm that oncolytic viruses act in a multimodal scheme, triggering lyses, immunogenic cell death and finally inducing anti-cancer immune responses. In this paper, we tested the local administration of a novel oncolytic adenovirus AdV-D24-ICOSL-CD40L expressing co-stimulatory molecules ICOSL and CD40L to induce the production of tumour infiltrating lymphocytes to the site of injection. Subsequently, in immunocompetent mouse models, we studied possible correlation between tumour infiltrates and anti-cancer efficacy. Described results showed that the delivery of oncolytic viruses encoding immunomodulatory transgenes in combination with anti-PD1 resulted in synergistic inhibition of both melanoma and mesothelioma tumours. Importantly anti-cancer effect positively correlated with cytotoxic CD8+ tumour-infiltrating lymphocytes exerting a central role in the tumour volume control thus generating beneficial outcomes that will undoubtedly provide new insights into possible future treatment strategies to combat cancer. Altogether our findings highlight the importance of oncolytic vectors able to modulate anti-cancer immune responses that can correlate with efficacy in solid malignancies.

Novel Insights Into Mesothelioma Therapy: Emerging Avenues and Future Prospects.

Malignant mesothelioma is a rare and aggressive cancer that develops in the thin layer surrounding the mesothelium and is mainly caused by asbestos exposure. Despite improvements in patient prognosis with conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, there are still no curative treatment modalities for advanced disease. In recent years, new therapeutic avenues have been explored. Improved understanding of the mechanisms underlying the dynamic tumor interaction with the immune system has led to the development of immunotherapeutic approaches. Numerous recent clinical trials have shown a desire to develop more effective treatments that can be used to fight against the disease. Immune checkpoint inhibitors, oncolytic adenoviruses, and their combination represent a promising strategy that can be used to synergistically overcome immunosuppression in the mesothelioma tumor microenvironment. This review provides a synthesized overview of the current state of knowledge on new therapeutic options for mesothelioma with a focus on the results of clinical trials conducted in the field.

The Multifaceted Roles of Mast Cells in Immune Homeostasis, Infections and Cancers.

Mast cells (MCs) play important roles in normal immune responses and pathological states. The location of MCs on the boundaries between tissues and the external environment, including gut mucosal surfaces, lungs, skin, and around blood vessels, suggests a multitude of immunological functions. Thus, MCs are pivotal for host defense against different antigens, including allergens and microbial pathogens. MCs can produce and respond to physiological mediators and chemokines to modulate inflammation. As long-lived, tissue-resident cells, MCs indeed mediate acute inflammatory responses such as those evident in allergic reactions. Furthermore, MCs participate in innate and adaptive immune responses to bacteria, viruses, fungi, and parasites. The control of MC activation or stabilization is a powerful tool in regulating tissue homeostasis and pathogen clearance. Moreover, MCs contribute to maintaining the homeostatic equilibrium between host and resident microbiota, and they engage in crosstalk between the resident and recruited hematopoietic cells. In this review, we provide a comprehensive overview of the functions of MCs in health and disease. Further, we discuss how mouse models of MC deficiency have become useful tools for establishing MCs as a potential cellular target for treating inflammatory disorders.

The Antifungal Action Mode of N-Phenacyldibromobenzimidazoles.

Our study aimed to characterise the action mode of N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure-activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e-f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action (5e with 2,4-Cl2 or 5f with 3,4-Cl2). The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC50 = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160-4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-ß-d-glucosaminide and 4-nitrophenyl-ß-d-N,N',N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing ß-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives.

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h-i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c-d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

Polymer Coated Oncolytic Adenovirus to Selectively Target Hepatocellular Carcinoma Cells.

Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.

Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model.

Malignant melanoma, an aggressive form of skin cancer, has a low five-year survival rate in patients with advanced disease. Immunotherapy represents a promising approach to improve survival rates among patients at advanced stage. Herein, the aim of the study was to design and produce, by using engineering tools, a novel oncolytic adenovirus AdV-D24- inducible co-stimulator ligand (ICOSL)-CD40L expressing potent co-stimulatory molecules enhancing clinical efficacy through the modulation of anti-cancer immune responses. Firstly, we demonstrated the vector's identity and genetic stability by restriction enzyme assay and sequencing, then, by performing in vitro and in vivo pre-clinical studies we explored the anti-cancer efficacy of the virus alone or in combination with anti PD-1 inhibitor in human melanoma cell lines, i.e., MUG Mel-1 and MUG Mel-2, and in immunocompetent C57BL/6 melanoma B16V mouse model. We showed that both monotherapy and combination approaches exhibit enhanced anti-cancer ability and immunogenic cell death in in vitro settings. Furthermore, AdV-D24-ICOSL-CD40L combined with anti PD-1 revealed a fall in tumor volume and 100% survival in in vivo context, thus suggesting enhanced efficacy and survival via complementary anti-cancer properties of those agents in melanoma therapy. Collectively, the novel oncolytic vector AdV-D24-ICOSL-CD40L alone or in combination with anticancer drugs, such as check point inhibitors, may open novel therapeutic perspectives for the treatment of melanoma.

Cancer-derived EVs show tropism for tissues at early stage of neoplastic transformation.

From the past decade, extracellular vesicles (EVs) have attracted considerable attention as tools for the selective delivery of anti-neoplastic drugs to cancer tissues. Compared to other nanoparticles, EVs display interesting unique features including immune compatibility, low toxicity and the ability to encapsulate a large variety of small- and macro-molecules. However, in virtually all studies, investigations on EVs have been focused on fully transformed cancers: the possibility to apply EV technology also to early-stage tumors has never been explored. Methods: Herein, we studied the ability of cancer-derived EVs to recognize and deliver their cargo also to incipient cancers. To this purpose, EV biodistribution was studied in MMTV-NeuT genetically modified mice during early mammary transformation, in fully developed breast tumors and in the normal gland of wild type syngeneic mice. EVs were loaded with indocyanine green (ICG), a near-infrared (NIR) dye together with oncolytic viruses and i.v. injected in mice. The nanoparticle biodistribution was assayed by in vivo and ex vivo optical imaging (detecting the ICG) and semiquantitative real-time PCR (measuring the adenoviral genome) in different tissues. Results: Our results demonstrate the ability of cancer-derived EVs to recognize early-stage neoplastic tissues opening the possibility to selectively deliver theranostics also for tumor prevention. Conclusions: Taken together our study demonstrates the ability of EVs to recognize and deliver diagnostic and therapeutic agents not only to fully transformed tissues but also to early stage tumors. These findings pave the way for the synthesis of "universal" EVs-based formulation for targeted cancer therapy.

From Conventional Therapies to Immunotherapy: Melanoma Treatment in Review.

In this review, we discuss the use of oncolytic viruses and checkpoint inhibitors in cancer immunotherapy in melanoma, with a particular focus on combinatory therapies. Oncolytic viruses are promising and novel anti-cancer agents, currently under investigation in many clinical trials both as monotherapy and in combination with other therapeutics. They have shown the ability to exhibit synergistic anticancer activity with checkpoint inhibitors, chemotherapy, radiotherapy. A coupling between oncolytic viruses and checkpoint inhibitors is a well-accepted strategy for future cancer therapies. However, eradicating advanced cancers and tailoring the immune response for complete tumor clearance is an ongoing problem. Despite current advances in cancer research, monotherapy has shown limited efficacy against solid tumors. Therefore, current improvements in virus targeting, genetic modification, enhanced immunogenicity, improved oncolytic properties and combination strategies have a potential to widen the applications of immuno-oncology (IO) in cancer treatment. Here, we summarize the strategy of combinatory therapy with an oncolytic vector to combat melanoma and highlight the need to optimize current practices and improve clinical outcomes.

Antifungal polybrominated proxyphylline derivative induces Candida albicans calcineurin stress response in Galleria mellonella.

Candida albicans CNB1 plays a role in the response in vitro and in vivo to stress generated by PB-WUT-01, namely 1,3-dimethyl-7-(2-((1-(3-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl)-1H-purine-2,6(3H,7H)-dione. The antifungal mechanism involved the calcineurin pathway-regulated genes SAP9-10. Galleria mellonella treated with PB-WUT-01 (at 0.64 µg/mg) showed limited candidiasis and remained within the highest survival rates. The molecular mode of action of PB-WUT-01 was rationalized by in silico docking studies toward both human and C. albicans calcineurin A (CNA) and calcineurin B (CNB) complexes, respectively. PB-WUT-01 acting as a calcineurin inhibitor in the C. albicans cells enhances the cells' susceptibility. Therefore it could be a suitable alternative treatment in patients with candidiasis.

Prospects of Replication-Deficient Adenovirus Based Vaccine Development against SARS-CoV-2.

The current appearance of the new SARS coronavirus 2 (SARS-CoV-2) and it quickly spreading across the world poses a global health emergency. The serious outbreak position is affecting people worldwide and requires rapid measures to be taken by healthcare systems and governments. Vaccinations represent the most effective strategy to prevent the epidemic of the virus and to further reduce morbidity and mortality with long-lasting effects. Nevertheless, currently there are no licensed vaccines for the novel coronaviruses. Researchers and clinicians from all over the world are advancing the development of a vaccine against novel human SARS-CoV-2 using various approaches. Herein, we aim to present and discuss the progress and prospects in the field of vaccine research towards SARS-CoV-2 using adenovirus (AdV) replication deficient-based strategies, with a comprehension that may support research and combat this recent world health emergency.

Sulfone derivatives enter the cytoplasm of Candida albicans sessile cells.

Since our study showed that sulfone derivatives' action mode creates a lesser risk of inducing widespread resistance among Candida spp., we continued verifying sulfones' antifungal activity using the following newly synthesized derivatives: bromodichloromethy-4-hydrazinyl-3-nitrophenyl sulfone (S1), difluoroiodomethyl-4-hydrazinyl-3-nitrophenyl sulfone (S2), and chlorodifluoromethyl-4-hydrazinyl-3-nitrophenyl sulfone (S3). As the mechanism by which sulfones gain access to the cytoplasm has not been elucidated yet, in order to track S1-3, we coupled their hydrazine group with BODIPY (final S1-3 BODIPY-labelled were named SB1-3). This approach allowed us to follow the vital internalization and endocytic routing of SB1-3, while BODIPY interacts primarily with fungal surfaces, thus confirming that S1-3 and their counterparts SB1-2 behaved as non-typical agents by damaging the cell membrane and wall after being endocytosed (SB1-3 fluorescence visible inside the unlysed sessile cells). Thus greatly decreasing the likelihood of the appearance of strains resistance. Core sulfones S1-3 are a promising alternative not only to treat planktonic C. albicans but also biofilm-embedded cells. In the flow cytometric analysis, the planktonic cell surface was digested by S1-3, which made the externalized PS accessible to AnnexinV binding and PI input (accidental cell death ACD). The occurrence of ACD as well as apoptosis (crescent-shaped nuclei) and anoikis of sessile cells (regulated cell death by 100%-reduction in attachment to epithelium) was assessed through monitoring the AO/PI/HO342 markers. CLSM revealed the invasion of S1-3 and SB1-3 in C. albicans without inducing cell lysis. This was a novel approach in which QCM-D was used for real-time in situ detection of viscoelastic changes in the C. albicans biofilm, and its interaction with S1 as a representative of the sulfones tested. S1 (not toxic in vivo) is a potent fungicidal agent against C. albicans and could be administered to treat invasive candidiasis as a monotherapy or in combination with antifungal agents of reference to treat C. albicans infections.