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Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Local pulmonary delivery of biotherapeutics may offer advantages for the treatment of lung diseases. Delivery of the therapeutic entity directly to the lung has the potential for a rapid onset of action, reduced systemic exposure and the need for a lower dose, as well as needleless administration. However, formulation of a protein for inhaled delivery is challenging and requires proteins with favorable biophysical properties suitable to withstand the forces associated with formulation, delivery, and inhalation devices. Nanobodies are the smallest functional fragments derived from a naturally occurring heavy chain-only immunoglobulin. They are highly soluble, stable, and show biophysical characteristics that are particularly well suited for pulmonary delivery. This paper highlights a number of clinical and preclinical studies on antibodies delivered via the pulmonary route and describes the advantages of using Nanobodies for inhaled delivery to the lung. The latter is illustrated by the specific example of ALX-0171, a Nanobody in clinical development for the treatment of respiratory syncytial virus (RSV) infections.

Local pulmonary delivery of biotherapeutics may offer advantages for the treatment of lung diseases. Delivery of the therapeutic entity directly to the lung has the potential for a rapid onset of action, reduced systemic exposure and the need for a lower dose, as well as needleless administration. However, formulation of a protein for inhaled delivery is challenging and requires proteins with favorable biophysical properties suitable to withstand the forces associated with formulation, delivery, and inhalation devices. Nanobodies are the smallest functional fragments derived from a naturally occurring heavy chain-only immunoglobulin. They are highly soluble, stable, and show biophysical characteristics that are particularly well suited for pulmonary delivery. This paper highlights a number of clinical and preclinical studies on antibodies delivered via the pulmonary route and describes the advantages of using Nanobodies for inhaled delivery to the lung. The latter is illustrated by the specific example of ALX-0171, a Nanobody in clinical development for the treatment of respiratory syncytial virus (RSV) infections.