Mariangela Garofalo

The Evolution of Adenoviral Vectors through Genetic and Chemical Surface Modifications

A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

The Anti-Proliferative Effect of L-Carnosine Correlates with a Decreased Expression of Hypoxia Inducible Factor 1 alpha in Human Colon Cancer Cells

In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS) production. In the present study we identified the Hypoxia-Inducible Factor 1α (HIF-1α) as a possible target of L-carnosine in HCT-116 cell line. HIF-1α protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1α induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1α. In the current study we found that L-carnosine reduces the HIF-1α protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1α degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively). Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1α expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1α activity has not been yet elucidated, this ability may be promising in treating hypoxia-related diseases.

Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma

ABSTRACT The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAds immunogenicity might be important for their efficacy. Therefore, we developed a versatile and rapid system to adsorb tumor-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response toward the tumor epitopes. By studying the model epitope SIINFEKL, we demonstrated that the peptide-coated OAd (PeptiCRAd) retains its infectivity and the cross presentation of the modified-exogenous epitope on MHC-I is not hindered. We then showed that the SIINFEKL-targeting PeptiCRAd achieves a superior antitumor efficacy and increases the percentage of antitumor CD8+ T cells and mature epitope-specific dendritic cells in vivo. PeptiCRAds loaded with clinically relevant tumor epitopes derived from tyrosinase-related protein 2 (TRP-2) and human gp100 could reduce the growth of primary-treated tumors and secondary-untreated melanomas, promoting the expansion of antigen-specific T-cell populations. Finally, we tested PeptiCRAd in humanized mice bearing human melanomas. In this model, a PeptiCRAd targeting the human melanoma-associated antigen A1 (MAGE-A1) and expressing granulocyte and macrophage colony-stimulating factor (GM-CSF) was able to eradicate established tumors and increased the human MAGE-A1-specific CD8+ T cell population. Herein, we show that the immunogenicity of OAds plays a key role in their efficacy and it can be exploited to direct the immune response system toward exogenous tumor epitopes. This versatile and rapid system overcomes the immunodominance of the virus and elicits a tumor-specific immune response, making PeptiCRAd a promising approach for clinical testing.

Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

Oncolytic Adenovirus Loaded with L-carnosine as Novel Strategy to Enhance the Antitumor Activity

Oncolytic viruses are able to specifically replicate, infect, and kill only cancer cells. Their combination with chemotherapeutic drugs has shown promising results due to the synergistic action of virus and drugs; the combinatorial therapy is considered a potential clinically relevant approach for cancer. In this study, we optimized a strategy to absorb peptides on the viral capsid, based on electrostatic interaction, and used this strategy to deliver an active antitumor drug. We used L-carnosine, a naturally occurring histidine dipeptide with a significant antiproliferative activity. An ad hoc modified, positively charged L-carnosine was combined with the capsid of an oncolytic adenovirus to generate an electrostatic virus–carnosine complex. This complex showed enhanced antitumor efficacy in vitro and in vivo in different tumor models. In HCT-116 colorectal and A549 lung cancer cell lines, the complex showed higher transduction ratio and infectious titer compared with an uncoated oncolytic adenovirus. The in vivo efficacy of the complex was tested in lung and colon cancer xenograft models, showing a significant reduction in tumor growth. Importantly, we investigated the molecular mechanisms underlying the effects of complex on tumor growth reduction. We found that complex induces apoptosis in both cell lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in future studies also for delivery of other bioactive drugs. Mol Cancer Ther; 15(4); 651–60. ©2016 AACR.

Synergistic anti‐tumor efficacy of immunogenic adenovirus ONCOS‐102 (Ad5/3‐D24‐GM‐CSF) and standard of care chemotherapy in preclinical mesothelioma model

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1‐year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed‐Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression‐free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS‐102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM‐CSF. The safety and immune activating properties of ONCOS‐102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS‐102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS‐102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti‐tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti‐tumor activity in the mesothelioma mouse model, ONCOS‐102 was able to slow down tumor growth. Interestingly, a synergistic anti‐tumor effect was seen when ONCOS‐102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS‐102 in combination with first‐line chemotherapy in patients suffering from malignant mesothelioma.

Isoflavones in aglycone solution enhance ultraviolet B-induced DNA damage repair efficiency

The isoflavones daidzein and genistein are natural compounds which have anti‐inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)‐induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)‐aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm2). For this purpose, human skin cells were first UVB‐irradiated and then treated with RPH‐aglycone. Comet assay analysis was used to estimate the UVB‐induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH‐aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34–35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB‐induced DNA damage.

Antitumor-specific T-cell responses induced by oncolytic adenovirus ONCOS-102 (AdV5/3-D24-GM-CSF) in peritoneal mesothelioma mouse model: KURYK et al.

Oncolytic adenoviral immunotherapy activates the innate immune system with subsequent induction of adaptive tumor‐specific immune responses to fight cancer. Hence, oncolytic viruses do not only eradicate cancer cells by direct lysis, but also generate antitumor immune response, allowing for long‐lasting cancer control and tumor reduction. Their therapeutic effect can be further enhanced by arming the oncolytic adenovirus with costimulatory transgenes and/or coadministration with other antitumor therapies. ONCOS‐102 has already been found to be well tolerated and efficacious against some types of treatment‐refractory tumors, including mesothelin‐positive ovarian cancer (NCT01598129). It induced local and systemic CD8+ T‐cell immunity and upregulated programmed death ligand 1. These results strongly advocate the use of ONCOS‐102 in combination with other therapeutic strategies in advanced and refractory tumors, especially those expressing the mesothelin antigen. The in vivo work presented herein describes the ability of the oncolytic adenovirus ONCOS‐102 to induce mesothelin‐specific T‐cells after the administration of the virus in bagg albino (BALB/c) mice with mesothelin‐positive tumors. We also demonstrate the effectiveness of the interferon‐γ the enzyme‐linked immunospot (ELISPOT) assay to detect the induction of T‐cells recognizing mesothelin, hexon, and E1A antigens in ONCOS‐102‐treated mesothelioma‐bearing BALB/c mice. Thus, the ELISPOT assay could be useful to monitor the progress of therapy with ONCOS‐102.

Antitumor effect of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment

&NA; Standard of care for cancer is commonly a combination of surgery with radiotherapy or chemoradiotherapy. However, in some advanced cancer patients this approach might still remaininefficient and may cause many side effects, including severe complications and even death. Oncolytic viruses exhibit different anti‐cancer mechanisms compared with conventional therapies, allowing the possibility for improved effect in cancer therapy. Chemotherapeutics combined with oncolytic viruses exhibit stronger cytotoxic responses and oncolysis. Here, we have investigated the systemic delivery of the oncolytic adenovirus and paclitaxel encapsulated in extracellular vesicles (EV) formulation that, in vitro, significantly increased the transduction ratio and the infectious titer when compared with the virus and paclitaxel alone. We demonstrated that the obtained EV formulation reduced the in vivo tumor growth in animal xenograft model of human lung cancer. Indeed, we found that combined treatment of oncolytic adenovirus and paclitaxel encapsulated in EV has enhanced anticancer effects both in vitro and in vivo in lung cancer models. Transcriptomic comparison carried out on the explanted xenografts from the different treatment groups revealed that only 5.3% of the differentially expressed genes were overlapping indicating that a de novo genetic program is triggered by the presence of the encapsulated paclitaxel: this novel genetic program might be responsible of the observed enhanced antitumor effect. Our work provides a promising approach combining anticancer drugs and viral therapies by intravenous EV delivery as a strategy for the lung cancer treatment. Graphical abstract Figure. No Caption available.

A novel in silico framework to improve MHC-I epitopes and break the tolerance to melanoma

ABSTRACT Tolerance toward tumor antigens, which are shared by normal tissues, have often limited the efficacy of cancer vaccines. However, wild type epitopes can be tweaked to activate cross-reactive T-cell clones, resulting in antitumor activity. The design of these analogs (i.e., heteroclitic peptides) can be difficult and time-consuming since no automated in silico tools are available. Hereby we describe the development of an in silico framework to improve the selection of heteroclitic peptides. The Epitope Discovery and Improvement System (EDIS) was first validated by studying the model antigen SIINFEKL. Based on artificial neural network (ANN) predictions, we selected two mutant analogs that are characterized by an increased MHC-I binding affinity (SIINFAKL) or increased TCR stimulation (SIIWFEKL). Therapeutic vaccination using optimized peptides resulted in enhanced antitumor activity and against B16.OVA melanomas in vivo. The translational potential of the EDIS platform was further demonstrated by studying the melanoma-associated antigen tyrosinase related protein 2 (TRP2). Following therapeutic immunization with the EDIS-derived epitope SVYDFFAWL, a significant reduction in the growth of established B16.F10 tumors was observed, suggesting a break in the tolerance toward the wild type epitope. Finally, we tested a multi vaccine approach, demonstrating that combination of wild type and mutant epitopes targeting both TRP2 and OVA antigens increases the antitumor response. In conclusion, by taking advantage of available prediction servers and molecular dynamics simulations, we generated an innovative platform for studying the initial sequences and selecting lead candidates with improved immunological features. Taken together, EDIS is the first automated algorithm-driven platform to speed up the design of heteroclitic peptides that can be publicly queried online.