Therapeutic extracellular vesicle production is substantially increased by inhibition of cellular cholesterol biosynthesis.

Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of the manufacturing process are rich with opportunities to enhance the ability to deliver these medicines to patients. With the goal of improving cell culture EV productivity, we have utilized high throughput siRNA screens to identify the underlying genetic pathways that regulate EV productivity to inform rational host cell line engineering and media development approaches. The screens identified multiple metabolic pathways of potential interest; one of which was validated and shown to be a ready implementable, cost-effective strategy to increase EV titers. We show that both EV volumetric and specific productivity from HEK293 and CHO-S were increased in a dose and cell line-dependent manner up to ninefold when cholesterol synthesis was inhibited by the inclusion of statins in the cell culture media. In addition, we show in response to statin treatment, elevation of EV markers in mesenchymal stem cell (MSC) cell culture media suggesting this approach can also be applicable to MSC EVs. Furthermore, we show that the EVs produced from statin-treated HEK293 cultures are effectively loaded by both endogenous and exogenous loading methods and have equivalent in vitro or in vivo potency relative to EVs from untreated cultures.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity.

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

A national survey of terrorism preparedness training among pediatric, family practice, and emergency medicine programs.

OBJECTIVES: Domestic terrorism is a real threat focusing on a need to engage in effective emergency preparedness planning and training. Front-line physicians are an important component of any emergency preparedness plan. Potential victims of an attack include children who have unique physiologic and psychological vulnerabilities in disasters. Front-line providers need to have adequate training to effectively participate in local planning initiatives and to recognize and treat casualties including children. The goal of the survey was to assess the current state of terrorism preparedness training, including child victims, by emergency medicine, family practice, and pediatric residency programs in the United States and to assess methods of training and barriers to establishing effective training. METHODS: A survey was e-mailed to a comprehensive list of all US pediatric, family practice, and emergency medicine residency programs 3 times between September 2003 and January 2004. The survey measured the perceived risk of terrorist attack, level of training by type of attack, level of training regarding children, method of training, and barriers to training. RESULTS: Overall, 21% of programs responded (46 of 182 pediatric, 75 of 400 family practice, and 29 of 125 emergency medicine programs). Across all of the event types, emergency medicine programs were more likely to report adequate/comprehensive training. However, < 50% of emergency medicine programs report adequate training for children. Didactic classroom-based lectures were the most commonly used method of training. Emergency medicine programs were more likely to use scenario-based exercises. Among programs that use scenario exercises, 93% report that they never (40%) or only sometimes (53%) incorporate child victims into the scenarios. Time, funding, access to subject matter experts, and availability of training material are the most important barriers to effective training. CONCLUSIONS: Children are a precious national resource and a vulnerable population in disasters. Despite the availability of terrorism preparedness funding, these data suggest that we are failing to provide adequate training to front-line providers who may care for children in a catastrophic domestic terrorist event.