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Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer., Cancer therapy using oncolytic virus has shown pre-clinical and clinical efficacy. Here, the authors report ExtraCRAd, an oncolytic virus cloaked with tumour cell membrane and report its therapeutic effects in vitro and in vivo in multiple mouse tumour models.

Background Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms. Methods The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids. Results Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations. Conclusion Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.

Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.

Virotherpay is emerging as a promising strategy against cancer, and three oncolytic viruses (OVs) have gained approval in different countries for the treatment of several cancer types. Beyond the capability to selectively infect, replicate and lyse cancer cells, OVs act through a multitude of events, including modification of the tumour micro/macro-environment as well as a complex modulation of the anti-tumour immune response by activation of danger signals and immunogenic cell death pathways. Most OVs show limited effects, depending on the viral platform and the interactions with the host. OVs used as monotherapy only in a minority of patients elicited a full response. Better outcomes were obtained using OVs in combination with other treatments, such as immune therapy or chemotherapy, suggesting that the full potential of OVs can be unleashed in combination with other treatment modalities. Here, we report the main described combination of OVs with conventional chemotherapeutic agents: platinum salts, mitotic inhibitors, anthracyclines and other antibiotics, anti-metabolites, alkylating agents and topoisomerase inhibitors. Additionally, our work provides an overview of OV combination with targeted therapies: histone deacetylase inhibitors, kinase inhibitors, monoclonal antibodies, inhibitors of DNA repair, inhibitors of the proteasome complex and statins that demonstrated enhanced OV anti-neoplastic activity. Although further studies are required to assess the best combinations to translate the results in the clinic, it is clear that combined therapies, acting with complementary mechanisms of action might be useful to target cancer lesions resistant to currently available treatments.

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (&gamma, PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using &gamma, PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into &gamma, PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

Background Attenuated measles virus (MV) strains are promising agents currently being tested against solid tumors or hematologic malignancies in ongoing phase I and II clinical trials; factors determining oncolytic virotherapy success remain poorly understood, however. Methods We performed RNA sequencing and gene set enrichment analysis to identify pathways differentially activated in MV-resistant (n = 3) and -permissive (n = 2) tumors derived from resected human glioblastoma (GBM) specimens and propagated as xenografts (PDX). Using a unique gene signature we identified, we generated a diagonal linear discriminant analysis (DLDA) classification algorithm to predict MV responders and nonresponders, which was validated in additional randomly selected GBM and ovarian cancer PDX and 10 GBM patients treated with MV in a phase I trial. GBM PDX lines were also treated with the US Food and Drug Administration-approved JAK inhibitor, ruxolitinib, for 48 hours prior to MV infection and virus production, STAT1/3 signaling and interferon stimulated gene expression was assessed. All statistical tests were two-sided. Results Constitutive interferon pathway activation, as reflected in the DLDA algorithm, was identified as the key determinant for MV replication, independent of virus receptor expression, in MV-permissive and -resistant GBM PDXs. Using these lines as the training data for the DLDA algorithm, we confirmed the accuracy of our algorithm in predicting MV response in randomly selected GBM PDX ovarian cancer PDXs. Using the DLDA prediction algorithm, we demonstrate that virus replication in patient tumors is inversely correlated with expression of this resistance gene signature (ρ = -0.717, P = .03). In vitro inhibition of the interferon response pathway with the JAK inhibitor ruxolitinib was able to overcome resistance and increase virus production (1000-fold, P = .03) in GBM PDX lines. Conclusions These findings document a key mechanism of tumor resistance to oncolytic MV therapy and describe for the first time the development of a prediction algorithm to preselect for oncolytic treatment or combinatorial strategies.

Activation of immune checkpoint pathways and limited T- cell infiltration result in immunological escape of tumors. Although immune checkpoint inhibitors are currently approved for several types of cancers, the response rate is often limited by the lack of tumor specific T-cells within the malignant tissue. Therefore, new combinatorial strategies are needed to enhance the clinical benefit of immune checkpoint inhibitors. We have previously developed PeptiCRAd, an oncolytic vaccine platform capable of directing the immune response toward tumor epitopes. In this study, we evaluated whether the platform could be used to increase the response rate to checkpoint inhibitors in both highly immunogenic and poorly immunogenic tumors, such as melanoma and triple negative breast cancer (TNBC). We report here that anti-PD-L1 therapy in combination with PeptiCRAd significantly reduced the growth of melanomas and increased the response rate to checkpoint inhibition. In fact, we registered a higher rate of complete responses among mice treated with the combination. This approach promoted the presence of non-exhausted antigen-specific T-cells within the tumor in comparison to anti-PD-L1 monotherapy. Furthermore, we found that targeting both MHC-I and II restricted tumor epitopes was necessary to decrease the growth of the poorly immunogenic TNBC model 4T1 and that combination with PD-L1 blockade increased the number of responders to checkpoint inhibition. Finally, the described strategy was validated in a translational in vitro model using HLA matched human PBMCs and tumor cell lines. Consistent to our previous results, improved cytotoxicity was observed with combination of PeptiCRAd and anti-PD-L1. These results demonstrate that oncolytic virus based cancer vaccine can significantly improve the response rate to checkpoint blocking antibodies in the context of immunogenic and non-immunogenic tumors.

Vaccinia virus has been studied extensively since its discovery as a smallpox vaccine in 1798. Its use as a smallpox vaccine documented its safety profile. It was later found that its large size and ability to accept large fragments of DNA combined with its natural tumor affinity make it an attractive agent for cancer therapy. This chapter discusses the history of the vaccinia, the various strains available, the biology of the virus as well as the steps in creating recombinants. The various clinical and safety considerations will be addressed. We will also discuss the various methods used to treat cancer using the vaccinia virus and will review the recent clinical trials using vaccinia in the treatment of cancer.

Purpose: Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a “serial treatment” consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM–CSF)–coding capsid chimeric adenovirus, CGTG-102. Results: Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking. Conclusions: These results provide the first human data linking antiviral immunity with antitumor immunity, implying that oncolytic viruses could have an important role in cancer immunotherapy. Clin Cancer Res; 19(10); 2734–44. ©2013 AACR.

Oncolytic viruses represent a multifaceted tool for cancer treatment. In addition to specific killing of cancer cells (oncolysis), these agents also provide danger signals prompting the immune system to stimulate an antitumor immune response. To increase adenovirus adjuvancy, we engineered the genome of Ad5D24 by inserting 18 immunostimulatory islands (Ad5D24-CpG). The toxicity and immunogenicity profile of Ad5D24-CpG showed that the safety of the maternal virus was retained. The efficacy of the CpG-enriched virus was assessed in a xenograft model of lung cancer where a significant increase in antitumor effect was seen in comparison with controls. When the experiment was repeated in animal depleted of natural killer (NK) cells, Ad5D24-CpG lost its advantage. The same was seen when Toll-like receptor (TLR)9 was blocked systemically. In a syngeneic model of melanoma (B16-OVA), we observed a significant increase of OVA-specific T cells and a decrease of activation of myeloid-derived suppressor cells in Ad5D24-CpG–treated mice. In conclusion, we have generated the first genetically modified oncolytic adenovirus backbone able to enhance TLR9-stimulation for increased antitumor activity.

Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (TH1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)–mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the TH1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8+ T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of TH1 cytokines. Cancer Res; 72(9); 2327–38. ©2012 AACR.

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.

BackgroundCancer stem cells/initiating cells (CSC/CIC), are thought to exist as a small population in malignant tissues. They are resistant to conventional cancer treatments and possibly underlie post-treatment relapse. The CIC population can be targeted with capsid modified oncolytic adenoviruses.Methodology/principal findingsWe studied the mechanisms of innate immunity to oncolytic adenovirus Ad5/3-Delta24 in conventional treatment resistant non-CIC breast cancer cells, breast cancer CD44(+)/CD24(-/low) CIC population and normal breast tissue CD44(+)/CD24(-/low) stem cells. We compared virus recognition by pattern recognition receptors for adenovirus, Toll-like receptors (TLR) 2 and 9 and virus induced type I interferon (IFN) response regulation in these cell types. We show TLR mediated virus recognition in these non-immune cell types. Normal tissue stem cells have intact type I IFN signaling. Furthermore, TLR9 and TLR2 reside constantly in recognition sites, implying constant activation. In contrast, breast cancer CD44(+)/CD24(-/low) CIC have dysregulated innate immune responses featuring dysfunctional virus recognition caused by impaired trafficking of TLR9 and cofactor MyD88 and the absence of TLR2, having a deleterious impact on TLR pattern recognition receptor signaling. Furthermore, the CIC have increased inhibitory signaling via the suppressor of cytokine signaling/Tyro3/Axl/Mer receptor tyrosine kinase (SOCS/TAM) pathway. These defects in contribute to dysfunctional induction of type I IFN response in CIC and therefore permissivity to oncolytic adenovirus.Conclusions/significanceCICs may underlie the incurable nature of relapsed or metastatic cancers and are therefore an important target regarding diagnostic and prognostic aspects as well as treatment of the disease. This study addresses the mechanisms of innate infection immunity in stem cells deepening the understanding of stem cell biology and may benefit not only virotherapy but also immunotherapy in general.

Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8+ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I–restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II–restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti–PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. Significance: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one.