Background

Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs.

Methods

Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells.

Results

Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs.

Conclusion

We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

Background

Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII).

Methods

We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208).

Results

Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28.

Conclusion

Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.