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Background T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. Methods The mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumorbearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. Results Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 day

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

The use of oncolytic viruses forms an appealing approach for cancer treatment. On the one hand the viruses replicate in, and kill, tumor cells, leading to their intra-tumoral amplification. On the other hand the viral infection will activate virus-directed immune responses, and may trigger immune responses directed against tumor cells and tumor antigens. To date, a wide variety of oncolytic viruses is being developed for use in cancer treatment. While the development of oncolytic viruses has often been initiated by researchers in academia and other public institutions, a large majority of the final product development and the testing of these products in clinical trials is industry led. As a consequence relatively few pre-clinical and clinical studies evaluated different oncolytic viruses in

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7−CD5− to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7−CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.In this article, Mikkers a

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced S

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus' tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two-and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus' capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.