Nanobody Conjugated Extracellular Vesicle Vaccine for Adaptive Immune Therapy

Extracellular vesicles (EVs) which are membrane enclosed nanoparticles secreted by nearly all cell types as a means of intercellular communication exhibit potential as a drug-delivering nanocarrier in a variety of applications. EVs from HEK293SF-3F6 3D suspension cells were isolated through size-exclusion chromatography. During EV biogenesis, HEK293SF-3F6 cells were incubated with Ac₄ManNAz, which led to azide incorporation into the EV surface-membrane glycoproteins. A derivative of the STING agonist diABZI, with a DBCO-PEG₁₂ handle, was conjugated with to the surface of EVs using strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to the EV surface azides. The EV conjugates were characterized by nanoparticle tracking analysis (NTA) and western blot. An in vitro cell culture experiment was conducted in which murine bone marrow-derived dendritic cells (BMDCs) were treated with the EV-diABZI conjugates and relevant controls. Flow cytometry showed increased median fluorescence intensity (MFI) of the BMDC activation markers CD40, CD80, and CD86, as well as Class II Major Histocompatibility Complex (MHCII) following treatment with EV-diABZI conjugates. Using an enzyme-linked immunosorbent assay (ELISA) assay, we evaluated secretion of interferon-β and the TNF-α, inflammatory cytokines secreted following innate immune activation in BMDCs. We observed an increase in secretion of both cytokines following treatment with EV-diABZI conjugates. Flow cytometry was also used to show that BMDCs presented the Class I MHC-restricted ovalbumin (OVA) epitope SIINFEKL on their surface at a higher-level following treatment with EV-diABZI conjugates that had been engineered to package OVA into their EV lumens (EV-OVA-diABZI), meaning that the BMDCs most efficiently took up and presented OVA when packaged within an EV-diABZI conjugate. Another experiment was done in vivo using mice to determine if an antigen-specific T cell response could be generated following vaccination with EV-OVA-diABZI conjugates. Unfortunately, since diABZI is a potent inflammatory agent and the OVA to diABZI ratio was not yet optimized, there was not enough OVA in the EV-OVA-diABZI vaccine to mount a significant T cell response to vaccination as quantified using an H2-kb/SIINFEKL tetramer. Using Shuffle T7 E. coli, a camelid VHH nanobody targeting MHCII was produced. The nanobody was designed with a C-terminal sortag motif (LPETGGHHHHHHEPEA) to facilitate sortase-mediated conjugation of amine-containing small molecules to the C-terminus of the nanobody. We used this approach to label the nanobody with NH₂-PEG₃-N₃, followed by sulfo-Cy5-DBCO, to facilitate nanobody tracking. Flow cytometry showed that BMDC treatment with nMHCII-Cy5 demonstrated a profound increase in the MFI of the Cy5 channel compared to controls, suggesting that the nanobody was not only phagocytosed but also bound to the MHCII molecule presented on the BMDC surface. Future work will include ligating NH₂-PEG₃-BCN to nMHCII to facilitate nanobody conjugation to the surface of N₃-EVs to enhance EV targeting to antigen presenting cells (APCs). Additionally, diABZI loading will be optimized to facilitate appreciable OVA delivery without inducing diABZI-mediated toxicities using an EV-OVA-diABZI vaccine.