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 immunotherapy has revolutionized the way tumors are treated. Nevertheless, efficient and robust testing platforms are still missing, including clinically relevant human ex vivo tumor assays that allow pre-treatment testing of cancer therapies and selection of the most efficient and safe therapy for a specific patient. In the case of immunotherapy, this testing platform would require not only cancer cells but also the tumor microenvironment, including immune cells. Here we discuss the applications of patient-derived tumor organoid cultures and the possibilities in using complex Research.

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 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.

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Due to 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. While CD8+ anti-tumor 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 re-engaged 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 pre-immunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid-and tumorspecific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced anti-tumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen presenting cells (APCs). 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 anti-tumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD1, strengthening the rationale behind combination therapy with oncolytic viruses. Research.

Proliferative diabetic retinopathy (PDR) is a major diabetic microvascular complication characterized by pathological angiogenesis. Several retinopathy animal models have been developed to study the disease mechanisms and putative targets. However, knowledge on the human proliferative disease remains incomplete, relying on steady-state results from thin histological neovascular tissue sections and vitreous samples. New translational models are thus required to comprehensively understand the disease pathophysiology and develop improved therapeutic interventions. We describe here a clinically relevant model, whereby the native multicellular PDR landscape and neo(fibro)vascular processes can be analysed ex vivo and related to clinical data. As characterized by three-dimensional whole-mount immunofluorescence and electron microscopy, heterogeneity in patient-derived PDR neovascular tissues included discontinuous capillaries coupled with aberrantly differentiated, lymphatic-like and tortuous endothelia. Spatially confined apoptosis and proliferation coexisted with inflammatory cell infiltration and unique vascular islet formation. Ex vivo-cultured explants retained multicellularity, islet patterning and capillary or fibrotic outgrowth in response to vitreoretinal factors. Strikingly, PDR neovascular tissues, whose matched vitreous samples enhanced lymphatic endothelial cell sprouting, contained lymphatic-like capillaries in vivo and developed Prox1 capillaries and sprouts with lymphatic endothelial ultrastructures ex vivo. Among multiple vitreal components, vascular endothelial growth factor C was one factor found at lymphatic endothelium-activating concentrations. These results indicate that the ischaemia-induced and inflammation-induced human PDR microenvironment supports pathological neolymphovascularization, providing a new concept regarding PDR mechanisms and targeting options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2 + breast cancer cells likewise led to increased MST1/ 2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2 + MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2 + breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8 T cells in the tumor microenvironment can be significantly enhanced.