Accelerated and intensified manufacturing of an adenovirus-vectored vaccine to enable rapid outbreak response.

The Coalition for Epidemic Preparedness Innovations' "100-day moonshot" aspires to launch a new vaccine within 100 days of pathogen identification, followed by large-scale vaccine availability within the "second hundred days." Here, we describe work to optimize adenoviral vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing output from the available manufacturing footprint. We describe a rapid virus 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 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 approximately fourfold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10,000 doses from each liter of bioreactor capacity. 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.

Transcriptomic analysis reveals mode of action of butyric acid supplementation in an intensified CHO cell fed-batch process.

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.

Oncolytic viruses: adenoviruses.

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.

Triple Space-Time Yield in Discontinuous Antibody Biomanufacturing by Combination of Synergetic Process Intensification Strategies.

Monoclonal antibodies are the workhorse of the pharmaceutical industry due to their potential to treat a variety of different diseases while providing high specificity and efficiency. As a consequence, a variety of production processes have been established within the biomanufacturing industry. However, the rapidly increasing demand for therapeutic molecules amid the recent COVID-19 pandemic demonstrated that there still is a clear need to establish novel, highly productive, and flexible production processes. Within this work, we designed a novel discontinuous process by combining two intensification strategies, thus increasing inoculation density and media exchange via a fluidized bed centrifuge, to fulfill the need for a flexible and highly productive production process for therapeutic molecules. To establish this new process, firstly, a small-scale experiment was conducted to verify synergies between both intensification strategies, followed by a process transfer towards the proof-of-concept scale. The combination of these two-process intensification measures revealed overall synergies resulting in decreased process duration (-37%) and strongly enhanced product formation (+116%) in comparison to the not-intensified standard operation. This led to an impressive threefold increase in space-time yield, while only negligible differences in product quality could be observed. Overall, this novel process not only increases the ways to react to emergency situations thanks to its flexibility and possible short development times, but also represents a possible alternative to the current established processes due to high increases in productivity, in comparison to standard fed-batch operations.

Boosting Productivity for Advanced Biomanufacturing by Re-Using Viable Cells.

Monoclonal antibodies (mAb) have gained enormous therapeutic application during the last decade as highly efficient and flexible tools for the treatment of various diseases. Despite this success, there remain opportunities to drive down the manufacturing costs of antibody-based therapies through cost efficiency measures. To reduce production costs, novel process intensification methods based on state-of-the-art fed-batch and perfusion have been implemented during the last few years. Building on process intensification, we demonstrate the feasibility and benefits of a novel, innovative hybrid process that combines the robustness of a fed-batch operation with the benefits of a complete media exchange enabled through a fluidized bed centrifuge (FBC). In an initial small-scale FBC-mimic screening, we investigated multiple process parameters, resulting in increased cell proliferation and an elongated viability profile. Consecutively, the most productive process scenario was transferred to the 5-L scale, further optimized and compared to a standard fed-batch process. Our data show that the novel hybrid process enables significantly higher peak cell densities (163%) and an impressive increase in mAb amount of approximately 254% while utilizing the same reactor size and process duration of the standard fed-batch operation. Furthermore, our data show comparable critical quality attributes (CQAs) between the processes and reveal scale-up possibilities and no need for extensive additional process monitoring. Therefore, this novel process intensification strategy yields strong potential for transfer into future industrial manufacturing processes.

A novel hybrid bioprocess strategy addressing key challenges of advanced biomanufacturing.

Monoclonal antibodies (mAb) are commonly manufactured by either discontinuous operations like fed-batch (FB) or continuous processes such as steady-state perfusion. Both process types comprise opposing advantages and disadvantages in areas such as plant utilization, feasible cell densities, media consumption and process monitoring effort. In this study, we show feasibility of a promising novel hybrid process strategy that combines beneficial attributes of both process formats. In detail, our strategy comprises a short duration FB, followed by a fast media exchange and cell density readjustment, marking the start of the next FB cycle. Utilizing a small-scale screening tool, we were able to identify beneficial process parameters, including FB interval duration and reinoculation cell density, that allow for multiple cycles of the outlined process in a reproducible manner. In addition, we could demonstrate scalability of the process to a 5L benchtop system, using a fluidized-bed centrifuge as scalable media exchange system. The novel process showed increased productivity (+217%) as well as longer cultivation duration, in comparison to a standard FB with a significantly lower media consumption per produced product (-50%) and a decreased need for process monitoring, in comparison to a perfusion cultivation. Further, the process revealed constant glycosylation pattern in comparison to the perfusion cultivation and has strong potential for further scale-up, due to the use of fully scalable cultivation and media exchange platforms. In summary, we have developed a novel hybrid process strategy that tackles the key challenges of current biomanufacturing of either low productivity or high media consumption, representing a new and innovative approach for future process intensification efforts.

Rapid intensification of an established CHO cell fed-batch process.

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.

Tumor Targeting of Oncolytic Adenoviruses Using Bispecific Adapter Proteins.

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.

Live vaccines-a short-cut to cancer viro-immunotherapy.

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.

Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy.

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.

Targeting polysialic acid-abundant cancers using oncolytic adenoviruses with fibers fused to active bacteriophage borne endosialidase.

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.

Perioperative, Spatiotemporally Coordinated Activation of T and NK Cells Prevents Recurrence of Pancreatic Cancer.

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.

PolySia-Specific Retargeting of Oncolytic Viruses Triggers Tumor-Specific Immune Responses and Facilitates Therapy of Disseminated Lung Cancer.

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.