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Analysis of of activation markers on CD8 T cells after neoadjuvant treatment.

Figure Legends for Figure S1-S5

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and disseminating cancer resistant to therapy, including checkpoint immunotherapies, and early tumor resection and (neo)adjuvant chemotherapy fails to improve a poor prognosis. In a transgenic mouse model of resectable PDAC, we investigated the coordinated activation of T and natural killier (NK) cells in addition to gemcitabine chemotherapy to prevent tumor recurrence. Only neoadjuvant, but not adjuvant treatment with a PD-1 antagonist effectively supported chemotherapy and suppressed local tumor recurrence and improved survival involving both NK and T cells. Local T-cell activation was confirmed by increased tumor infiltration with CD103+CD8+ T cells and neoantigen-specific CD8 T lymphocytes against the marker neoepitope LAMA4-G1254V. To achieve effective prevention of distant metastases in a complementary approach, we blocked the NK-cell checkpoint CD96, an inhibitory NK-cell receptor that binds CD155, which was abundantly expressed in primary PDAC and metastases of human patients. In gemcitabine-treated mice, neoadjuvant PD-1 blockade followed by adjuvant inhibition of CD96 significantly prevented relapse of PDAC, allowing for long-term survival. In summary, our results show in an aggressively growing transgenic mouse model of PDAC that the coordinated activation of both innate and adaptive immunity can effectively reduce the risk of tumor recurrence after surgery, facilitating long-term remission of this lethal disease.Significance: Coordinated neoadjuvant and adjuvant immunotherapies reduce the risk of disease relapse after resection of murine PDAC, suggesting this concept for future clinical trials. Cancer Res; 78(2); 475–88. ©2017 AACR.

Process intensification is increasingly used in the mammalian biomanufacturing industry. The key driver of this trend is the need for more efficient and flexible production strategies to cope with the increased demand for biotherapeutics predicted in the next years. Therefore, such intensified production strategies should be designed, established, and characterized. We established a CHO cell process consisting of an intensified fed-batch (iFB), which is inoculated by an N-1 perfusion process that reaches high cell concentrations (100 × 106 c ml−1). We investigated the impact of butyric acid (BA) supplementation in this iFB process. Most prominently, higher cellular productivities of more than 33% were achieved, thus 3.5 g L−1 of immunoglobulin G (IgG) was produced in 6.5 days. Impacts on critical product quality attributes were small. To understand the biological mechanisms of BA in the iFB process, we performed a detailed transcriptomic analysis. Affected gene sets reflected concurrent inhibition of cell proliferation and impact on histone modification. These translate into subsequently enhanced mechanisms of protein biosynthesis: enriched regulation of transcription, messenger RNA processing and transport, ribosomal translation, and cellular trafficking of IgG intermediates. Furthermore, we identified mutual tackling points for optimization by gene engineering. The presented strategy can contribute to meet future requirements in the continuously demanding field of biotherapeutics production.

The Coalition for Epidemic Preparedness Innovations’ ‘100-day moonshot’ aspires to launch a new vaccine within 100 days of pathogen identification. Here, we describe work to optimize adenovirus vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing the output from the available manufacturing footprint.We describe a rapid viral seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a new perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by around four-fold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10000 doses from each liter of bioreactor capacity.Transition to a new production process across a large manufacturing network is a major task. In the short term, the rapid seed generation workflow could be used with the existing production process. We also use techno-economic modelling to show that, if linear scale-up were achieved, a single cleanroom containing two 2000 L bioreactors running our new perfusion-based process could supply bulk drug substance for around 120 million doses each month, costing This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.

Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as economic as possible. However, intensified processes often require more development time compared with conventional fed-batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N-stage FB by implementing N-1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step-wise improved and intensified rapidly in multi-parallel small-scale bioreactors using N-1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell-specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.

Tumour immunotherapies have been a breakthrough in clinical oncology but only a few patients benefit from this progress. Additional interventions that sensitize immunologically cold tumours for the administration of checkpoint modifiers are urgently needed. In this issue of EMBO Molecular Medicine, Aznar et al present the already approved yellow fever vaccine 17D as an oncolytic agent for tumour immunoactivation. In tumour-bearing mice, they demonstrated a convincing synergy of the vaccine with CD137 agonistic antibodies resulting in significantly improved survival.

Tumor-selectively replicating (oncolytic) viruses are promising tools for therapy of solid cancers and have been initially developed to achieve potent tumor lysis with acceptable side effects on healthy tissue. However, in recent years, oncolytic viruses have been recognized as therapeutic vehicles exhibiting multipronged anti-tumoral activity. Apart from direct cytolysis, stimulation of both innate and adaptive tumor-directed immune responses have been recognized as important mechanisms of oncolytic virotherapy, which were probably decisive in achieving the long-term tumor remissions that oncolytic viruses have shown in clinical trials in advanced melanoma. In this short review, we will introduce basic mechanisms of viral oncolysis and the current state of clinical development. With a focus on oncolytic adenoviruses, we will describe the efforts to restrict oncolytic virus infection to tumor tissue using conditional replication and targeted delivery. Furthermore, we will discuss ways to optimize virus-mediated immunostimulation and the potential of virotherapy as an integrative part of systemic tumor immunotherapies.

Tumor-selectively replicating "oncolytic" adenoviruses based on serotype 5 are promising tools for the treatment of solid tumors. However, their effective delivery to the tumor by systemic administration remains challenging. Several strategies of molecular retargeting have been pursued to equip adenoviruses with molecular features that facilitate their efficient uptake by tumors and to protect healthy tissue from damage. Transductional retargeting can be conveniently achieved using bispecific molecular adapter proteins based on the ectodomain of the coxsackievirus and adenovirus receptor linked to tumor ligands of choice. In this chapter, we describe methods for their design, purification, and application.

Tumor-selectively replicating "oncolytic" adenoviruses based on serotype 5 are promising tools for the treatment of solid tumors. However, their effective delivery to the tumor by systemic administration remains challenging. Several strategies of molecular retargeting have been pursued to equip adenoviruses with molecular features that facilitate their efficient uptake by tumors and to protect healthy tissue from damage. Transductional retargeting can be conveniently achieved using bispecific molecular adapter proteins based on the ectodomain of the coxsackievirus and adenovirus receptor linked to tumor ligands of choice. In this chapter, we describe methods for their design, purification, and application.

Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice. The therapeutic benefit achieved by tumor retargeting of antiviral antibodies is largely due to NK cell-mediated triggering of tumor-directed CD8 T-cells. We further demonstrate that antibody-retargeting (Ab-retargeting) is a feasible method to sensitize tumors to PD-1 immune checkpoint blockade. In therapeutic settings, Ab-retargeting greatly improves the outcome of intratumor application of an oncolytic adenovirus and facilitates long-term survival in treated animals when combined with PD-1 checkpoint inhibition. Tumor-directed retargeting of preexisting or virotherapy-induced antiviral antibodies therefore represents a promising strategy to fully exploit the immunotherapeutic potential of oncolytic virotherapy and checkpoint inhibition., The efficacy of oncolytic adenoviruses is limited by strong immune responses being induced against the oncolytic virus itself. Here, the authors generate a bispecific molecule capable of redirecting the adenovirus-specific antibodies to tumour cells and show this induces immune mediated cancer growth inhibition and enhances the therapeutic efficacy of viral oncolysis.

In this study, we examined the portrayal of privacy and data protection on social network sites (SNS) in German media. As a widely used social web application, SNS arouse a considerable amount of media attention. By means of a value-framing analysis, the present study investigated how German media frame privacy and data protection on SNS. In a first step, the three value frames informational self-determination, security and psychosocial need satisfaction were identified through a qualitative framing analysis of online media. In a second step, we conducted a standardized content analysis of four preselected German newspapers to quantify the previously identified frames. Results show that newspapers mainly frame SNS in matters of users’ informational self-determination and that they predominantly present SNS as a threat to this value.

Genetic replacement of adenoviral fiber knobs by ligands that enable tumor specific targeting of oncolytic adenoviruses is challenging because the fiber knob contributes to virus assembly. Here, we present a novel concept by describing stable recombinant adenoviruses with tumor specific infection mode. The fiber knob was replaced by endosialidaseNF (endoNF), the tailspike protein of bacteriophage K1F. EndoNF recognizes polysialic acid, an oncofetal antigen characteristic for high malignant tumors of neuroendocrine origin. An intramolecular chaperone contained in endoNF warrants folding and compensates for the knob function in virus assembly. Obtained recombinant viruses demonstrated polysialic acid dependent infection modes, strong oncolytic capacity with polysialic acid positive cells in culture and a high potential to inhibit tumor growth in a therapeutic mouse model of subcutaneous neuroblastoma. With a single genetic manipulation we achieved ablation of the fiber knob, introduction of a tumor specific ligand, and folding control over the chimeric fiber construct.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and disseminating cancer resistant to therapy, including checkpoint immunotherapies, and early tumor resection and (neo)adjuvant chemotherapy fails to improve a poor prognosis. In a transgenic mouse model of resectable PDAC, we investigated the coordinated activation of T and natural killier (NK) cells in addition to gemcitabine chemotherapy to prevent tumor recurrence. Only neoadjuvant, but not adjuvant treatment with a PD-1 antagonist effectively supported chemotherapy and suppressed local tumor recurrence and improved survival involving both NK and T cells. Local T-cell activation was confirmed by increased tumor infiltration with CD103+CD8+ T cells and neoantigen-specific CD8 T lymphocytes against the marker neoepitope LAMA4-G1254V. To achieve effective prevention of distant metastases in a complementary approach, we blocked the NK-cell checkpoint CD96, an inhibitory NK-cell receptor that binds CD155, which was abundantly expressed in primary PDAC and metastases of human patients. In gemcitabine-treated mice, neoadjuvant PD-1 blockade followed by adjuvant inhibition of CD96 significantly prevented relapse of PDAC, allowing for long-term survival. In summary, our results show in an aggressively growing transgenic mouse model of PDAC that the coordinated activation of both innate and adaptive immunity can effectively reduce the risk of tumor recurrence after surgery, facilitating long-term remission of this lethal disease. Significance: Coordinated neoadjuvant and adjuvant immunotherapies reduce the risk of disease relapse after resection of murine PDAC, suggesting this concept for future clinical trials. Cancer Res; 78(2); 475–88. ©2017 AACR.

Polysialic acid (polySia) is expressed on several malignant tumors of neuroendocrine origin, including small cell lung cancer. In this study, we investigated the therapeutic efficacy of tumor-directed T-cell responses, elicited by polySia-retargeted oncolytic adenovirus infection, in an orthotopic murine model of disseminated polySia-positive lung cancer. In several cell lines, we demonstrated highly polySia-selective retargeting of adenoviral infection using a bispecific adapter comprising the ectodomain of the coxsackievirus/adenovirus receptor and a polySia-recognizing single-chain antibody domain. PolySia-dependent systemic infection in vivo facilitated effective uptake of viruses in subcutaneous polySia-expressing human tumors, whereas hepatic viral load and hepatotoxicity were significantly reduced. The impact and nature of antitumoral immune responses triggered by systemic delivery of polySia-retargeted oncolytic adenoviruses were investigated in an orthotopic model of disseminated lung cancer. Interestingly, improved transduction by polySia-retargeted oncolytic adenoviruses led to CD45-positive cell infiltrates in close association with large lytic areas. Consistently, enhanced tumor regression and prolonged survival was only observed in immunocompetent mice, but not in T-cell–deficient mice. To investigate whether improved systemic infection by polySia retargeting would elicit a tumor-specific T-cell response, we screened the used lung cancer cells for mutated oncogenes by complete exon sequencing. In agreement with our other results, only retargeted oncolysis was able to induce a significant response specific for the tumor-associated neoepitope Gsta2-Y9H. In conclusion, we demonstrated that effective retargeting of oncolytic adenovirus against polySia-expressing tumors elicits an effective tumor-directed T-cell response after systemic virus delivery and facilitates therapy of disseminated lung cancer. Cancer Immunol Res; 3(7); 751–63. ©2015 AACR.

Today, head-up displays (HUDs) are commonly used in cars to show basic driving information in the visual field of the viewer. This allows information to be perceived in a quick and easy to understand manner. With advances in technology, HUDs will allow richer information to be conveyed to the driver by exploiting the third dimension. We envision a stereoscopic HUD for displaying content in 3D space. This requires an understanding of how parallaxes impact the user's performance and comfort, which is the focus of this work. In two user studies, involving 49 participants, we (a) gather insights into how projection distances and stereoscopic visualizations influence the comfort zone and (b) the depth judgment of the user. The results show that with larger projection distances both the comfort zone and the minimum comfortable viewing distance increase. Higher distances between the viewer and a real world object to be judged decrease the judgment accuracy.

This study investigated if cognitive skills, mood, attitudes and personality traits influence quality perceptions, modality choice (speech vs. touch), and performance. It was shown that attitudes and mood are related to quality perceptions while performance is linked to personality traits. Modality choice is influenced by attitudes and personality. Cognitive abilities had no effect.

International audience; Mobile devices have conquered our daily life. They are carried and used at almost all times and in any situation sometimes even against legal restriction - e.g. in the car. Services running on these devices (e.g. email, text messages, etc.) include graphical (GUI) and voice interface (VUI) causing visual distraction for the driver although they could solely be operated by speech in- and output. As a result services should adapt their interfaces due to the specific usage scenario. The aim of this work is therefore to develop design recommendations based on a cognitive model for the voice user interface taking the particularities of the specific scenario (e.g. reduction of off-road eye gazes) into account. We assume that distraction is mainly due to an increased effort and a decreased expectancy of the VUI compared to the GUI. Design recommendations derived from these considerations will be described by means of a concrete example.