Andrea Vitale

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.

Phenotypic and genetic characterization of a family carrying two Xq21.1-21.3 interstitial deletions associated with syndromic hearing loss

BackgroundSensorineural hearing impairment is a common pathological manifestation in patients affected by X-linked intellectual disability. A few cases of interstitial deletions at Xq21 with several different phenotypic characteristics have been described, but to date, a complete molecular characterization of the deletions harboring disease-causing genes is still missing. Thus, the aim of this study is to realize a detailed clinical and molecular analysis of a family affected by syndromic X-linked hearing loss with intellectual disability.ResultsClinical analyses revealed a very complex phenotype that included inner ear malformations, vestibular problems, choroideremia and hypotonia with a peculiar pattern of phenotypic variability. Genomic analysis revealed, for the first time, the presence of two close interstitial deletions in the Xq21.1-21.3, harboring 11 protein coding, 9 non-coding genes and 19 pseudogenes. Among these, 3 protein coding genes have already been associated with X-linked hearing loss, intellectual disability and choroideremia.ConclusionsIn this study we highlighted the presence of peculiar genotypic and phenotypic details in a family affected by syndromic X-linked hearing loss with intellectual disability. We identified two, previously unreported, Xq21.1-21.3 interstitial deletions. The two rearrangements, containing several genes, segregate with the clinical features, suggesting their role in the pathogenicity. However, not all the observed phenotypic features can be clearly associated with the known genes thus, further study is necessary to determine regions involved.

3q29 microduplication in a small family with complex metabolic phenotype from Southern Italy

aAndrea Vitale and Giuseppe Labruna contributed equally to this work. *Corresponding authors: Pasqualina Buono, Ceinge Biotecnologie Avanzate, Via G. Salvatore 486, 80145, Naples, Italy, Phone: 00390813737892, E-mail: buono@uniparthenope.it; Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy; and IRCCS SDN, Naples, Italy; and Barbara Lombardo, Ceinge Biotecnologie Avanzate, Via G. Salvatore 486, 80145, Naples, Italy, Phone: 00390813737917, E-mail: lombardo@ceinge.unina.it; and Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Andrea Vitale, Annamaria Mancini and Andreina Alfieri: Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy; and Ceinge Biotecnologie Avanzate, Naples, Italy Giuseppe Labruna: IRCCS SDN, Naples, Italy Laura Iaffaldano and Carmela Nardelli: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Fabrizio Pasanisi: Dipartimento di Medicina Clinica e Chirurgia, Università Federico II, Naples, Italy Lucio Pastore: Ceinge Biotecnologie Avanzate, Naples, Italy; and Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Letter to the Editor

659. Oncolytic Adenovirus Loaded with Bioactive Modified Peptide as a Novel Approach to Treat Cancer

Cancer is still a leading cause of death worldwide. Although many kinds of treatment have been developed during the past decades, there is still a lack of effective therapy for advanced cancer. Currently treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase patient life expectancy. Therefore new treatment strategies against cancer are in high demand. Efficient anticancer agent and its targeted delivery into the tumor mass is a key prerequisite for the successful cancer therapy. Oncolytic virotherapy is emerging as a potential approach to treat cancer, using viruses, which are specifically engineered to selectively infect, replicate in and kill cancer cells without causing damage to normal cells. Their combination with chemotherapeutic agents have shown promising results due to the synergistic effect of viruses and drugs; therefore the combinatorial therapy is considered a beneficial approach for cancer treatment. Taken into account these considerations we optimized a strategy to conjugate peptides on the viral capsid, based on electrostatic interaction and used this strategy to deliver an active anti-tumor dipeptide. We used L-carnosine, a naturally occurring histidine dipeptide with anti-proliferative activity. A modified L-carnosine, positively charged was absorbed onto the viral capsid of an oncolytic adenovirus to generate a virus-carnosine complex. The complex showed enhanced anti tumor efficacy in vitro and in vivo and higher infectious titer compared to a naked oncolytic adenovirus in colorectal and lung cancer cells. The in vivo efficacy of the complex was analyzed in lung and colon cancer xenograft models, displaying a significant reduction in tumor growth and synergistic effect between virus and dipeptide. Moreover, we studied the molecular mechanisms underlying the effects of complex on tumor growth reduction. Complex can induce apoptosis in both cells lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in further studies also for specific delivery of other active drugs.

622. Oncolytic Adenoviruses Loaded With Active Drugs as a Novel Drug Delivery System for Cancer Therapy

L-carnosine (β-Ala-His) is a naturally occurring histidine dipeptide, normally found in brain, kidney and in large amounts in muscle. L-carnosine has biological functions, including antioxidant activity, ability to chelate metal ions, as well as anti-inflammatory and anti-senescence properties. Recent studies have demonstrated that 50-100 mM of L-carnosine decreases cell proliferation in a colon cancer cell line HCT116, bearing a mutation in codon 13 of the RAS proto-oncogene. In addition, pre-treatment with L-carnosine decreases the intracellular concentration of Adenosine Triphosphate (ATP) and Reactive Oxygen Species (ROS) and inhibits the cell cycle progression in the G1 phase. The proto-oncogene KRAS is mutated in a wide array of human cancers and is important both in tumour progression and resistance to anticancer drugs. To overcome treatment limitations due to the high intracellular concentration required we have hypothesized that L-carnosine can be conjugated on the capsid of oncolytic viruses. Oncolytic viruses are viruses that are able to replicate specifically in and destroy tumor cells and this property is either inherent or genetically-engineered. The association of viruses with specific drugs, would increase the efficacy of the treatment of human neoplasia due to the synergistic action of virus and drug. First we have developed a strategy to conjugate peptides on viral capsid, based on electrostatic interaction. Then, using different cancer cell lines we found that oncolytic virus coated with L-carnosine with a tail of positively charged polylysine was able to enhance a positive anticancer synergistic effect. Finally, in order to investigate the molecular mechanisms underlying the effect of tumor reduction by oncolytic virus coated with modified L-carnosine, we have used three different approaches. First, we have examined, in samples with virus alone, or in combination with L-carnosine, the oncolytic replication by evaluating the E1A expression, second the apoptotic mechanism by expression of specific genes and at end the autophagy regulation via the amount of LC3-II. In conclusion, we have developed a model to use oncolytic adenovirus as a scaffold to deliver active drugs. Once validated the proposed model could be used as a novel drug delivery system for cancer therapy.

220. Evaluation of the Efficacy of a New Oncolytic Vaccine Platform in Humanized Mice

Cancer immunotherapy represents a promising approach for the treatment of malignancies. However, breaking the immunosuppressive micro-environment is still a difficult but necessary condition to improve modern therapies. Oncolytic Adenoviruses (OAds) are able to elicit some degree of anti-tumor response and we are now investigating how OAds can be turned in novel cancer-vaccine platforms by exploiting their natural immunogenicity. The key feature of our peptide-coated conditionally-replicating adenoviruses (PeptiCRAd) is the physical conjugation of the adjuvant (i.e. OAds) to the tumor epitopes, in order to achieve a better co-delivery to antigen presenting cells (APCs).We coated the negatively charged OAds with a positive lysine-extended version of the major histocompatibility complex (MHC) class-I model epitope SIINFEKL (polyK-SIIN), and we observed no significant changes in the oncolytic activity compared to naked viruses. Then, we confirmed the cross presentation on MHC-I of the modified polyK-SIIN in absence or presence of the virus. Afterwards, using B16-OVA tumor-bearing immunecompetent mice, we compared the administration of polyK-SIIN-coated OAds (PeptiCRAd) to the administration of an OAds-SIINFEKL mix solution. Mice treated with PeptiCRAd showed smaller tumor volumes and higher levels of OVA-specific CD8+ T-lymphocytes compared to all control groups. Interestingly, we observed a strong (negative) correlation between the size of the tumors and the anti-OVA immune response. Moreover, we evaluated whether or not the different activation of dendritic cells (DCs) could explain the advantage of PeptiCRAd over the OAds-SIINFEKL mix: consistent with our hypothesis, PeptiCRAd promoted the expansion of mature (CD86+) and SIINFEKL-cross presenting CD11c+ DCs. Then we tested our PeptiCRAd technology using a multipeptide vaccine approach to evaluate if a broader targeting could improve the anti-tumor immune response. In addition, by monitoring the effect of the treatment on uninjected tumors we were also able to assess the effect of PeptiCRAd on metastasis.Finally, to collect human-relevant data, PeptiCrad efficacy, specificity and immunogenicity were assessed in humanized mice bearing implanted human melanomas.

Oncolytic adenoviruses coated with MHC-I tumor epitopes for a new oncolytic vaccine platform.

Meeting abstracts Oncolytic adenoviruses (OAds) demonstrated great potential for the treatment of cancer in many pre-clinical studies. The pre-existing immunity and their immunodominance, however, still limit the success of oncolytic virotherapy in cancer patients. Thus, a transition from oncolytic

Two novel genomic rearrangements identified in suicide subjects using a-CGH array

Many genes expressed in the central nervous system are candidates for increased susceptibility to suicidal behavior.... Language: en