Antibody Drug

ExoIL-12 from Codiak BioSciences is the first engineered clinically exosome therapeutic candidate in the world. Codiak obtained high purity exosomes by density gradient centrifugation, and used proteomic analyses on purified EVs, identifying two protein families previously unexplored as EV scaffolds, the EWI immunoglobulin superfamily (IGSF8 and PTGFRN) and the MARCKS protein family (MARCKS, MARKCSL1, and BASP1). Members of both protein families were found to be abundant in EVs derived from a variety of cell types and were selected for further investigation as scaffold proteins for EV loading. These scaffold proteins can be genetically modified to attach various biomolecules or drugs, such as cytokines, antibody fragments, RNA binding proteins, vaccine antigens, etc., which makes them a ideal platform for drug delivery. 

Researchers assessed the relative ability of these proteins to direct fusion partners into EVs using FLAG-tagged GFP as a surrogate cargo molecule and have attempted to quantitatively analyse them using traditional flow cytometery and ELISA methods. However, the traditional cytometry only qualitatively demonstrate that these proteins are expressed at the cellular level, cannot prove whether the target protein is effectively packaged into EVs. Meanwhile, ELISA can only determine that a protein is overexpressed but not whether this overexpression is prevalent in EVs or abounds in a subpopulation. Therefore, it is necessary to analyse EVs at the single particle level. Using the Flow NanoAnalzyer to identify scaffold proteins, and analyze the expression of several scaffold proteins at single EVs particle level, the researchers showed that PTGFRN protein was expressed in 97% of exosomes. In contrast to the semi-quantitative Western Blot and the ELISA method which can only provide an average value, the Flow NanoAnalzyer can determine the proportion of positive subpopulations. Furthermore, the fluorescence intensity can be used to further reflect information on the amount of protein expression on single EVs particle.

Figure 1. The expression of PTGFRN proteins and IL2

Figure 2. Proteins’s different proportion of expression on the Flow NanoAnalyzer

The application of the Flow NanoAnalzyer is the first single particle assay for genetically modified PTGFRN exosomes at single particle level. Traditional ELISA can only quantify the protein on a large number of exosomes, while the Flow NanoAnalzyer can identify and quantify the protein expression at the individual exosome level, and found that PTGFRN is an ideal scaffold protein with expression percentage of 97%.

Molecular Therapy, 2021, 29(5), 1729-1743.