Antibody conjugated nano and microparticle systems for targeted immunosuppression and purification of extracellular vesicles

Abstract

Helper T cell activity is dysregulated in a number of diseases with limited treatment options, including those associated with rheumatic autoimmunity. Phosphodiesterase 4 (PDE4) inhibition has the potential to enable controlled immunosuppression, but a targeted therapy has not yet been developed to leverage this opportunity. Inappropriate activation of the epidermal growth factor receptor (EGFR) is a driver of tumorigenesis in many cancers, such as colorectal cancer (CRC). The activation status of EGFR is reflected in subpopulations of extracellular vesicles (EVs) originating from cancer cells. However, current laboratory-based EV purification techniques are labor intensive and inefficient. This research addresses challenges in two distinct areas that are unified through the implementation of antibody conjugates that provide novel solutions for each challenge. The overall goals of this work are to enable both targeted immunosuppression of CD4+ T cells and the purification of EGFR+ cancer associated EVs. In this work we employed maleimide-thiol mediated conjugation of anti-CD4 F(ab’) antibody fragments to enable nanoparticle targeted delivery of Eggmanone, a specific PDE4 inhibitor, to CD4+ T cells. We also developed cetuximab-DNA conjugates with cleavable DNA linkers containing two unique restriction sites flanking a fluorescently modified residue to provide selective labeling of EGFR+ EVs purified with superparamagnetic microparticles. Our novel delivery system targeted the majority of CD4+ T cells present among a complex cell population, and we have demonstrated antigen specific inhibition of CD4+ T cell responses mediated by nanoparticle formulated Egm. Cetuximab decorated, superparamagnetic microparticles enabled the purification of EGFR+ EVs from conditioned media, and we demonstrated non-destructive release and fluorescent labeling of purified EVs. This work is the first characterization of Egm’s immunomodulatory potential, and it suggests the potential benefit of a biodegradable delivery vehicle that is designed for targeted immunosuppression. It is also the first characterization of a restriction enzyme cleavable antibody conjugate linker that enables non-destructive release of captured EVs and demonstrates the benefit of selective fluorescent labeling of purified EVs that could improve downstream analysis.