Immunological boosting and personalization of oncolytic virotherapies for cancer treatment

Cancer is the leading cause of death worldwide creating a need for novel cancer treatments that are more efficient but also safer and more specific. Oncolytic viruses (OVs) have shown a solid safety profile in clinical trials. OVs are nowadays considered immunotherapies because of to their ability to stimulate the host immune system to fight against cancer. Promising efficacy has been seen in some trials, however, efficacy is often seen only in a small group of patients. The purpose of the thesis was to improve the efficacy of OV therapies by boosting the immunogenicity of the viruses, and to optimize the therapeutic efficacy by selecting favorable patient populations and by developing a method to tailor the drug individually for each patient. In the first study, an oncolytic adenovirus (OAd) was modified to express human tumor necrosis factor alpha (hTNFα), a potent immunomodulatory cytokine. The TNFα-virus showed effective tumor cell killing associated with signs of immunogenic cell death and enhanced recruitment of immune cells to the infection site. We also saw potential for combining the TNFα-virus therapy with radiation. In another study the immunogenicity of an oncolytic vaccinia virus was enhanced by modifying it to express DNA-dependent activator of interferon-regulatory factors (DAI), a potent inducer of innate immune responses during virus infection. We showed that the DAI-virus induces expression of genes involved in immune responses, and treatments with the virus showed improved cancer-killing efficacy and immunogenicity in murine and human melanoma models, suggesting applicability also in vaccine design. Response rates after virotherapies vary between patients, and there is a lack of markers that would help predict the patient cohorts who would benefit from the therapy. We screened over 200 cancer patients treated with OAds for two Fc gamma receptor (FcγR) polymorphisms to determine if these polymorphisms would affect the responsiveness to the treatments. We observed a certain FcγR genotype combination (FcγRIIIa-VV + FcγRIIa-HR) to be predictive of poor overall survival after OAd treatments. To tailor the OV therapy for enhanced specificity, we developed a novel platform (PeptiCRAd) to coat a virus with tumor-specific antigens (peptides) for improved induction of cancer-specific immunity. Efficacy and immunogenic potency of the PeptiCRAd were shown in several in vivo models. Our results suggest that administration of tumor-specific peptides on the surface of OVs increases the anti-tumor efficacy compared to treatments with viruses or peptides alone. This platform has potential to be used as a carrier and adjuvant for patient-specific peptides to trigger anti-tumor immunity in a personalized manner.Syöpä on maailman yleisin kuolinsyy. Tarve uusille, tehokkaammille, turvallisemmille ja kohdistetummille syöpähoidoille on suuri. Onkolyyttisten (syöpäsoluja tappavien) virusten turvallisuus on osoitettu useissa kliinisissä tutkimuksissa. Onkolyyttisia virushoitoja pidetään nykyään yhtenä immunoterapian muotona, sillä niillä on kyky herättää immuunijärjestelmä taistelemaan syöpää vastaan. Nämä virushoidot ovat osoittaneet lupaavia tuloksia joissakin tutkimuksissa, mutta ne tehoavat usein vain pieneen joukkoon potilaita. Väitöskirjan tutkimusten tarkoituksena oli parantaa virushoitojen tehokkuutta lisäämällä virusten immunogeenisyyttä eli kykyä aktivoida kehon immuunipuolustus syöpäkasvainta vastaan. Lisäksi tavoitteena oli optimoida hoitojen tehokkuutta etsimällä hoidolle suotuisia potilasryhmiä sekä kehittää menetelmä, jolla virushoito voidaan räätälöidä yksilöllisesti kullekin potilaalle. Muunsimme geneettisesti onkolyyttisen adenoviruksen ja vaccinia-viruksen (lehmärokkoviruksen) tuottamaan immunomodulatorisia proteiineja kasvaimissa. Adenoviruksen muunsimme tuottamaan tuumorinekroositekijä alfa (TNFα) -sytokiinia, ja vaccinia-viruksen muunsimme tuottamaan DNA:ta tunnistavaa reseptoria, DAI:ta. Kumpikin uusi virus osoittautui tutkimuksissa tehokkaaksi syöpäsolujen tappajaksi ja sai aikaan tehostetun immuunipuolustuksen syöpää vastaan koe-eläimissä. Virushoitojen hoitovasteen on huomattu vaihtelevan suuresti potilaiden kesken. Toistaiseksi ei myöskään ole olemassa biomarkkereita, joiden avulla voitaisiin valikoida virushoidoista parhaiten hyötyvät potilaat. Määritimme yli 200:n onkolyyttisilla adenoviruksilla hoidetun syöpäpotilaan genotyypit kahden Fc gamma reseptoripolymorfian (FcyR) suhteen, ja tutkimme vaikuttavatko nämä polymorfismit potilaiden hoitovasteeseen. Havaitsimme tietyn FcyR genotyyppiyhdistelmän (FcγRIIIa-VV + FcyRIIa-HR) indikoivan huonoa elinajanodotetta, eli kyseisen genotyypin omaavat potilaat eivät näyttäisi hyötyvän virushoidoista. Syöpien monimuotoisuudesta ja potilaiden eroista johtuen yksi syöpähoito ei tehoa kaikkiin kasvaimiin. Siksi kehitimme uuden viruspohjaisen alustan (PeptiCRAd:n), jolla voidaan saada aikaan tarkennetumpi immuunivaste kasvainta vastaan. PeptiCRAd on kasvainspesifisillä peptideillä päällystetty onkolyyttinen virus, ikään kuin virus kasvaimen vaatteissa , jossa virusvektorin tehokkuus ja kyky herättää immuunivaste yhdistyy peptidihoitojen spesifiyteen. Osoitimme PeptiCRAd:n tehon ja immunogeenisyyden useissa kasvainmalleissa. Tuloksemme viittaavat siihen, että tällainen viruksen ja kasvainpeptidien yhdistelmä voisi toimia tehokkaana alustana personoidulle syöpähoidolle

Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

There is an errata corrige made after the publication.Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, allowing researchers to modify the tropism and biodistribution profile of the vector, leading to the production of viral vectors able to target different tissues and organs. This review describes recent non-genetic modifications of the surfaces of viral vectors to decrease immune system activation and to control tissue targeting. The developments described herein provide opportunities for applications of gene therapy to treat acquired disorders and genetic diseases and to become useful tools in regenerative medicine.Peer reviewe

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

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

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.Peer reviewe

Fc-gamma receptor polymorphisms as predictive and prognostic factors in patients receiving oncolytic adenovirus treatment

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

The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAd´s 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 towards 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 anti-tumor efficacy and increases the percentage of anti-tumor 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 towards 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.Peer reviewe

Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

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