Strategies to enhance efficacy of oncolytic virotherapy

Despite major advances in conventional cancer treatments by surgery, chemotherapy, radiotherapy and their combination, the outcome remains partially ineffective against numerous cancer types, for example mesothelioma, lung cancer, and colon cancer. Furthermore, due to resistance factors and the subsequent loss of response, which may occur rapidly during the conventional treatments regimes, new anti-cancer agents, presenting new mechanisms of action and lacking cross-resistance to commonly used therapies, are in high demand. Oncolytic virotherapy is a promising anti-cancer strategy, and the approval of the first oncolytic virus, Imlygic (T-Vec, talimogene laherparepvec), in Western world by US Food and Drug Administration (FDA) and European Medicines Agency (EMA) has opened up new perspectives for improved treatment of cancer. Single therapy is rarely successful in treating cancer, particularly in metastatic or advanced cancer, and survival rates with monotherapies alone are generally poor. The combination of multiple therapies to treat cancer has already shown significant results in the standard care of cancer. This strategy utilizes the combination of both conventional and novel therapies that can bring the future promise of cancer treatment. In this thesis it has been hypothesized that by combining oncolytic adenoviruses (oAd) with chemotherapeutic drugs and a biological agent we could improve anti-cancer efficacy through synergistic effect against cancer. Therefore, we have tested various treatment regimes with the overall goal being the improvement of oncolytic virotherapy efficacy. Secondly, since safety issues concerning gene therapy and viral vectors are tremendously important, we have performed studies on safety issues of adenoviral vectors. In brief, we have evaluated the anti-cancer activity of combination treatment with standard of care (SoC) chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and Ad5/3-d24-GM-CSF (ONCOS-102) in vitro and in a xenograft BALB/c model of human malignant mesothelioma (MM). We could show improved anti-tumor effects when ONCOS-102 was combined with SoC chemotherapy regimens over chemotherapy and virus alone. Combination therapy resulted in synergistic anti-cancer effect improving the therapeutic outcome. In a subsequent study we tested anti-cancer properties of the dipeptide L-Carnosine complexed with an oncolytic adenovirus (virus-L-Carnosine complex). The complex demonstrated improved anti-tumor efficacy both in vitro and in vivo in tested cancer models. In HCT116 colon and A549 lung cancer cells, the virus-L-Carnosine complex presented a higher transduction level and infectious titer over uncoated oncolytic adenovirus. The in vivo efficacy of the virus-L-Carnosine complex was tested in two cancer models: i) lung and ii) colon cancer xenograft mice models. It exhibited a significant reduction in tumor growth compared to other tested groups. Additionally, we investigated the molecular mechanism underlying the effects of the complex on tumor growth reduction. Safety assessment of viral vectors was performed in animal studies. Extensive studies on toxicity and bio-distribution of ONCOS-102 in Syrian hamsters and experiments in BALB/c nude mice indicated no side effects of repeated administration of oncolytic adenovirus. The side effects were evaluated by assessment of body weight, food consumption, hematology, clinical chemistry, histopathology and bio-distribution. We concluded that combinatory studies utilizing oncolytic viruses with standard of care chemotherapy and an experimental virus-L-Carnosine complex showed synergistic anti-cancer efficacy, thus providing a strong rationale for clinical testing of such combinations in mesothelioma, lung and colon cancer. Additionally, our studies suggested that adenovirus could be used in future studies for delivery of other bioactive drugs as a novel strategy in cancer therapy.Perinteiset syöpähoidot eli leikkaushoito, sädehoito, kemoterapia tai näiden yhdistelmät, ovat kehittyneet merkittävästi. Tästä huolimatta hoidon teho voi olla heikko tietyissä syöpätyypeissä kuten esimerkiksi mesotelioomassa, keuhkosyövässä tai paksusuolen syövässä. Lisäksi hoidon tehoa voi heikentää syövän kehittämä resistenssi käytettyä hoitomuotoa vastaan, jolloin saavutettu hoitovaste voidaan menettää. Tämän takia syövän uusien hoitomuotojen kehitys tärkeää. Erityisen tärkeää on kehittää hoitoja, joilla on uusi toimintamekanismi ja joihin syövän kehittämä hoitoresistenssi muita hoitomuotoja kohtaan ei vaikuta. Onkolyyttinen virushoito on eräs lupaava syövän hoitomuoto. Ensimmäinen länsimaissa hyväksytty onkolyyttinen virus on Imlygic (myös tunnettu nimillä T-Vec ja talimogene laherparepvec). Imlygicin hyväksyntä sekä USA:n (FDA) että Euroopan (EMA) lääkeviranomaisten toimesta on avannut uusia mahdollisuuksia syöpähoitojen kehittämisessä. Yhden hoitomuodon käyttö syövän hoidossa on harvoin tehokasta, erityisesti silloin kun kysessä on etäpesäkkeitä muodostava tai pitkälle edennyt tauti. Useiden hoitomuotojen yhdistäminen syövän hoidossa on osoittautunut merkittävästi tehokkaammaksi kuin vain yhden hoitomuodon käyttö. Tulevaisuudessa konventionaalisten ja uusien hoitomenetelmien yhdistäminen saattaa mahdollistaa merkittävästi parempien hoitotulosten saavuttamisen. Tässä väitöskirjassa on pyritty osoittaamaan, että yhdistämällä onkolyyttinen adenovirus kemoterapian tai biologisen yhdisteen kanssa, voidaan syövän hoitotehoa parantaa edellä mainittujen hoitojen yhteisvaikutuksen takia. Väitöskirjassa on kokoeiltu erilaisia hoitokombinaatioita, joiden tarkoituksena on ollut parantaa onkolyyttisen virushoidon tehokkuutta. Lisäksi väitöskirjassa on tutkittu adenovirusten turvallisuustekijöitä, koska geeninsiirron ja virusvektoreiden käytön turvallisuus on yleisesti erittäin tärkeää. Väitöskirjassa on tutkittu kemoterapiaan perustuvan käypähoidon (Pemetrexed, Cisplatin, Carboplatin) ja onkolyyttisen adenoviruksen Ad5/3-d24-GM-CSF (ONCOS-102) yhdistelmähoidon tehoa ihmisen malignin mesoteliooman (MM) malleissa, mitkä olivat erilaiset in vitro-mallit että BALB/c-ksenograftimalli. Tutkimuksessa osoitettiin, että hoidon teho käytetyissä malleissa parani, kun ONCOS-102 yhdistettiin käypähoidon kanssa verrattaessa pelkään virus- tai käypähoitoon. Yhdistelmähoito johti yhteisvaikutukseen, joka paransi hoidon tehoa. Väitöskirjassa tutkittiin hoitotehoa myös yhdistelmällä, missä dipeptidi L-karnosiinin oli kompleksoitu onkolyyttisen adenoviruksen kanssa (virus-L-karnosiinikompleksi). Kompleksin käyttö lisäsi tehoa käytetyissä in vitro- ja in vivo-syöpämalleissa. HCT116 paksusyöpäsolu- ja A549 keuhkosyöpäsolulinjoissa virus-L-karnosiinikompleksin käyttö tehosti viruksen transduktiota sekä lisäsi viruksen infektiivistä tiitteriä verrattuna virukseen jota ei oltu kompleksoitu. Virus-L-karnosiinikompleksia tutkittiin kahdessa in vivo-mallissa: keuhkosyövän ja paksusuolensyövän ksenograftihiirimalleissa. Kompleksin käyttö johti huomattavasti vähentyneeseen kasvaimen kasvuun verrattuna muihin käytettyihin hoitoryhmiin. Lisäksi väitöskirjassa tutkittiin kompleksin molekulaarista toimintamekanismia. Virusvektoreihin liittyvä turvallisuusarviointi tehtiin käyttämällä eläinkokeita. ONCOS-102-viruksen toksisuus- ja biodistribuutiotutkimukset Syyrian hamstereissa ja BALB/c-hiirimallissa eivät indikoineet viruksen toistuvan annostelun aiheuttavan sivuvaikutuksia. Sivuvaikutuksia arvioinnissa käytettiin eläimen painoa, ruuan kulutusta, hematotologiaa, kliinistä kemiaa, histopatologiaa sekä viruksen biodistribuutiota. Väitöskirjan tulosten perusteella onkolyyttisen viruksen yhdistäminen kemoterapiakäypähoitoon tai viruksen kompleksoiminen L-karnosiinin kanssa johtavat syöpähoidon tehoa parantavaan yhdistelmävaikutukseen. Tämä antaa vahvan perusteen tutkia yhdistelmien tehoa mesoteliooman sekä keuhko- ja paksusuolensyövän hoidossa potilailla. Lisäksi väitöskirjassa esitetyt tulokset viittaavat siihen, että adenovirusta voitaisiin käyttää muiden bioaktiivisten lääkkeiden kuljettamiseen kohteeseensa. Tämä olisi avian uusia strategia syövän hoidossa

34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 2

Document type: Articl

408 oncolytic vaccines in combination with pd l1 blockade for the treatment of melanoma

The Immunological escape of tumors represents one of the main obstacles to the treatment of malignancies. The approval of drugs able to disrupt the immune suppressive pathways through anti-CTLA-4 monoclonal antibodies represented a milestone in the history of immunotherapy. However, treatment with these immune checkpoint inhibitors (ICIs) seems to be effective only in small cohorts of patients. It has been proposed that the efficacy of ICIs relies on the presence of an undergoing immunological response. For this reason, we hypothesized that oncolytic vaccines, able to elicit a tumor specific response, would synergize with anti-PD-L1 therapy. B16 murine melanomas were established in immunocompetent C57 mice. Then mice were treated with anti-PD-L1 monotherapy, PeptiCRAd (oncolytic vaccine) monotherapy or a combination of the two. The growth of the tumors was analyzed. At the end of the experiment, all the mice mice were euthanized and organs collected for immunological analysis. We investigated antigen-specific T-cell responses and immune suppressive background by flow cytometry and ELISPOT assays

Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm2 (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 × 109 ± 0.2 and 1.75 × 109 ± 0.08 infectious particles of ONCOS-401 per cm2 of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses

Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm2 (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 × 109 ± 0.2 and 1.75 × 109 ± 0.08 infectious particles of ONCOS-401 per cm2 of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses

Heterologous and cross-species tropism of cancer-derived extracellular vesicles

Extracellular vesicles (EVs) are naturally occurring cargo delivery vesicles that have recently received considerable attention for their roles in intercellular communication in many physiological and pathological processes, including tumourigenesis. EVs generated by different tissues demonstrated specific homing: in particular, cancer-derived EVs showed a selective tropism for the tumor tissue from which the vesicles originated. For this property, EVs have been proposed as drug delivery tools for anti-cancer therapies, although the limited knowledge about their in vivo tropism hinders their therapeutic applications. The current study aimed to characterize the targeting properties of cancer- derived EVs in vitro and their biodistribution in vivo, by using an imaging approach. Methods. EVs were generated from: i) murine lung (LL/2) and colon (MC-38) cancer lines, ii) human lung cancer cell line (A549) and iii) human liver biopsy samples from healthy individuals. EVs were loaded with fluorescent dyes alone or in combination with a biopharmaceutical agent, the oncolytic adenovirus (OV), characterized for charge and size and tested for their activity in cancer cell lines. Finally, optical imaging was extensively applied to study in vivo and ex vivo the biodistribution of EVs originated from different sources in different mouse models of cancer, including xenograft, syngeneic graft and the MMTV-NeuT genetically modified animal. Results. We initially demonstrated that even loading EVs even with a large biopharmaceutical oncolytic viruses (OVs) did not significantly change their charge and dimension properties, while increasing their anti-neoplastic activity compared to the virus or EVs alone. Interestingly, this activity was observed even if the EVs derived from lung cancer were applied to colon carcinoma cell lines and vice versa, suggesting that the EV uptake occurred in vitro without any specificity for the cancer cells from which the vesicles originated. When administered i.v (intravenously) to the mouse models of cancer, the tumour-derived EVs, but not the EVs derived from a healthy tissue, demonstrated a selective accumulation of the fluorescence at the tumour site 24 h after injection; adding OVs to the formulation also did not change the tumour-specific tropism of the EVs also in vivo. Most interestingly, the in vivo experiments confirmed the in vitro observation of the generalized tropism of tumour-derived EVs for any neoplastic tissue, independent of the tumour type or even the species originating the vesicles. Conclusions. Taken together, our in vitro and in vivo data demonstrate for the first time a heterologous, cross-species tumour-tropism for cancer-derived EVs. This finding challenges our current view on the homing properties of EVs and opens new avenues for the selective delivery of diagnostic/therapeutic agents to solid tumours

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

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

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

Toxicological and bio-distribution profile of a GM-CSF-expressing, double-targeted, chimeric oncolytic adenovirus ONCOS-102-Support for clinical studies on advanced cancer treatment

The purpose of this work was to carry out preclinical toxicity and bio-distribution studies required for regulatory approval of a clinical trial application for Phase I clinical studies of ONCOS-102 (Ad5/3-D24-GM-CSF) for therapy of advanced cancers (NCT01598129). The study design, route of administration and dosage differs from the clinical protocol and in more detail, investigate bio-distribution and toxicological profile of ONCOS-102 treatment in animal model. The study was carried out in 300 hamsters divided into nine test groups-three bio-distribution groups and six groups for analysis of toxicity. Hamsters received ONCOS-102 by intracardial, intraperitoneal or subcutaneous injections. Additionally, one group was administered twice a week with intraperitoneal injections of Cyclophosphamide. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. No adverse effects of repeated administration of ONCOS-102 including body weight, food consumption, hematology and clinical chemistry parameters, histopathology and bio-accumulation were observed in the course of 6-month administration and following 3-month recovery period. All obtained findings indicate the treatment clinically safe.Peer reviewe