Abstract
A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.
Highlights
A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs)
The two VHHs are separated by a (GGGGS)[3] flexible spacer, and a myc-tag was added to the N′-terminus of glycophorin A (GPA)-VHH-based neutralizing agents (VNAs) and the C′-terminus of Kell-VNA to simplify analysis
Each red blood cells (RBCs) was estimated to express ~4,600,000 copies of GPA-VNA and 2,200,000 copies of Kell-VNA proteins per cell (Supplementary Fig. 1b; see Methods section and legend to Supplementary Fig. 1b for calculation). In this in vitro mouse fetal liver culture system, the VNA-expressing cells undergo enucleation at a level similar to control cells and have similar CD71 and Ter[119] surface expression, proliferation, and morphology compared to control cells, suggesting that these modifications do not disturb normal red cell differentiation (Supplementary Figs. 1c–f)
Summary
A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. Other groups have administered a combination of biotinylated VHH anti-BoNT/A and a fusion protein consisting of a scFv-specific to murine glycophorin A (GPA) and streptavidin This strategy increases the VHH retention time in the circulation as well as its neutralization potency in mice. We suggest that similar types of engineered human RBCs can be used to provide long-term protection against exposures to a variety of bacterial toxins and harmful viruses
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
