(I-343) Covalently Binding Programmed Cell Death Ligand-1 to Mesenchymal Stromal Cell Derived Extracellular Vesicles for the Treatment of Ocular Autoimmune Disease

Autoimmune uveitis is a sight-threatening condition and a major cause of visual disability worldwide. Current treatments require the use of corticosteroids and immune suppressive drugs which carry the risk of significant side effects. The key pathological feature of autoimmune uveitis is cytotoxic destruction of ocular tissues by Th1 and Th16 cells. Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) show promise in preclinical and early stage clinical trials for treating ocular autoimmune diseases because of their ability to suppress the activation of Th1 and Th17 cells.¹ Programmed death ligand-1 (PD-L1) is a well-studied immune check point inhibitor, also shown to directly suppress T cell activation and proliferation. While MSC-EVs and PD-L1 based therapies alone have potential for treating ocular autoimmune diseases, we hypothesize that combining PD-L1 with MSC-EVs will result in more potent treatment than either alone. Here we present a method to covalently tether dibenzocyclooctyne (DBCO) modified recombinant PD-L1 (DBCO-PDL1) to azide-EVs via strain promoted azide alkyne cycloaddition (SPAAC) to enhance the potency of MSC-EV treatment for ocular autoimmune diseases.

Induced pluripotent cell derived MSCs (ihMSCs) were cultured in vertical wheel bioreactors on spherical GelMA microcarriers. Azides were incorporated into EVs by treating cultures with 600 µM L-azidohomoalanine (AHA), an azide-containing analog of methionine, approximately 48 hrs prior to 70% confluency (approximately day 7 of culture). Next, the media was replaced with chemically defined protein free media and the cells were allowed to secrete azide-containing EVs into the media for 24 hrs. The EVs were recovered by filtration and ultracentrifugation and the size and yield of EVs were quantified by nanoparticle tracking analysis. To verify that AHA was successfully incorporated into the cells and their EVs, both were exposed to DBCO-FITC dye and analyzed by fluorescence microscopy or flow cytometry. Recombinant PD-L1 was functionalized with the strained cyclooctyne, DBCO, via EDC/NHS coupling reaction and purified by dialysis. To bind the DBCO-PDL1 to azide-EVs, the reagents were allowed to undergo a SPAAC click reaction under physiological conditions for 2 hrs. Bead-based flow cytometry was employed to verify the SPAAC reaction between DBCO-PDL1 and azide-EVs.

Staining with DBCO-FITC indicated that AHA was successfully incorporated into cells after 48 hr treatment (Fig 1A). The cell yield and viability (~95%) between control cultures and AHA treated cultures (Fig 1B-C) showed that AHA treated cells did not lose their viability or proliferative potential. Furthermore the total protein secreted into the media and total EV yield and size were also not reduced by AHA treatment (Fig 1D-F). When exposed to DBCO-FITC dye and analyzed via bead-based flow cytometry, AHA-EVs showed a shift in fluorescence compared to plain EVs (Fig 1G). These results together suggest that AHA incorporation did not interfere with ihMSC growth or protein synthesis, and that the AHA present in the EVs can readily partake in the SPAAC reaction. The modification of PD-L1 with DBCO via EDC/NHS coupling was completed with an efficiency of approximately 15% of available binding sites. The DBCO-PDL1 was then reacted with azide-EVs, captured on CD63 beads and counterstained for recombinant PD-L1 which indicated approximately 5% of the azide-EVs were successfully conjugated with PD-L1.

We have shown that ihMSC-EVs can be produced with azide functional groups which  can readily partake in SPAAC reactions. These results demonstrate the feasibility of covalently tethering recombinant proteins to EVs to potentially enhance their therapeutic potency. Furthermore, the EVs were produced in scalable suspension bioreactors with MSCs derived from induced pluripotent cells, a theoretically limitless supply of genetically identical cells, making this work a step towards GelMA microcarriers withmeeting the demand for large scale production of potent therapeutic EVs for immune-related diseases. Future work will focus on improving the efficiency of the SPAAC reaction and the effect of PDL1-MSC-EVs in treating experimental autoimmune uveitis.

1. Oh & Lee, Progress in Retinal and Eye Research, 2021


2. Fransisco et al., Immunology Review, 2010