Undergraduate Researcher University of Minnesota, Twin Cities, United States
Introduction/Rationale: Exosomal tetraspanins (EXTs) are four-pass transmembrane proteins enriched in exosomes and central regulators of exosome biogenesis. EXTs have potential as scaffolds for exosome engineering and vaccine design. Here, we develop EXT-based strategies that include (1) a generalizable approach for loading heterologous type I membrane proteins into exosomes using non-covalent leucine-zipper heterodimerization, and (2) a neoantigen-displaying exosome cancer vaccine.
Methods: Project 1: EXT family members (CD63/Y235A, TSPAN2/3/7, DMTsp39D) were fused to SynZip1 and co-expressed with SynZip2–tagged type I membrane proteins (CD4, CD43) in HEK293 cells. Exosomes were isolated by ultracentrifugation, and protein loading was assessed by immunoblotting (anti-HA, anti-Myc, CD9, HSP90).
Project 2: A mutant KRas epitope was inserted into the second extracellular loop of CD63/Y235A in three configurations. The optimal construct incorporated a flexible 2A linker to enhance folding and surface display. Constructs were expressed in CD63-knockout HEK293 cells, exosomes were purified by SEC, and loading and surface display were analyzed by immunoblotting (anti-2A, CD9, HSP90) and flow cytometry.
Results: In Project 1, all EXT–SynZip1 constructs recruited SynZip2-tagged cargo to exosomes. Among the combinations tested, TSPAN3–CD43 showed the highest exosome enrichment relative to the cell. In Project 2, flow cytometry revealed strong surface expression of both KRasG12D (99.7%) and CD63 (99.5%) in transfected CD63KO cells, with no detectable signal in untransfected controls. These results indicate that CD63/Y235A can serve as an effective scaffold for the exosomal display of tumor-specific antigens.
Conclusion: Concluding the studies performed, exosomal tetraspanins provide versatile platforms for exosome engineering. SynZip-mediated recruitment enables efficient membrane protein loading, while direct epitope fusion supports tumor antigen display, establishing a foundation for exosome-based vaccines and therapeutics.
