Extracellular vesicles for immunotherapy and tumor profiling

Extracellular vesicles are particles secreted by cells and mediators of intercellular communication, carrying a diverse cargo of lipids, nucleic acids, proteins and metabolites. The specific cargo and function of EVs depends on the cell of origin. EVs from dendritic cells (DC) loaded with tumor antigens induced anti-tumor CD8+ T cell responses and reduced tumor growth in mice. Studies have reported that EVs from human and cow's milk have anti-inflammatory effects and promote barrier functions. EVs from cancer cells can be detected in biofluids and may be useful tools for cancer diagnosis, prognosis and disease monitoring.

Adequate T cell priming and infiltration into the tumor improve the efficacy of PD- 1- and PD-L1-targeted immune checkpoint blockade ICB). Therefore, T cell responses generated by antigen-loaded (Ag-loaded) DC EVs have the potential to enhance the response to PD-1 and PD-L1 blockade. In addition, T cell responses to Ag-loaded EVs are dependent on B cells. In study I, we investigated a combination of Ag-loaded DC EVs with ICB of PD-1 or PD-L1. Therapeutically administered EVs did not improve the response to ICB in mice, but prophylactic administration of EVs strongly increased the survival of mice receiving therapeutic PD-L1 blockade. In study II, we used a fusion protein to target EVs specifically to B cells through human CD21. This targeting increased the Ag-specific CD8+ T cell expansion, which can indicate an increased anti-tumor potential.

In study II, we explored the use of bovine milk-derived EVs for antigen delivery and Ag-specific immunomodulation. EVs from bovine serum were studied in comparison. We achieved loading of the model antigen ovalbumin (OVA) into EVs, and these EVs increased OVA-specific CD4+ T cell proliferation compared to free OVA in vitro and in vivo. In vitro, OVA-loaded serum EVs induced a different phenotype of activated CD4+ T cells compared to OVA-loaded milk EVs or free OVA.

In study IV, we analyzed the protein content of urinary EVs from urinary bladder cancer (UBC) patients and non-UBC controls using proximity extension assay. We identified a UBC-specific signature and were able to distinguish muscle invasive UBC compared to non-muscle-invasive UBC, supporting EVs as biomarkers.

In conclusion, this thesis increases our understanding of the therapeutic and diagnostic potential of EVs.

List of scientific papers

I. Rosanne E. Veerman, Gozde Gucluler Akpinar, Annemarijn Offens, Loïc Steiner, Pia Larssen, Andreas Lundqvist, Mikael C.I. Karlsson, Susanne Gabrielsson. Antigen-loaded extracellular vesicles induce responsiveness to anti-PD-1 and anti-PD-L1 treatment in a checkpoint refractory melanoma model. Cancer Immunology Research. 11 217-227 (2023). https://doi.org/10.1158/2326-6066.cir-22-0540

II. Annemarijn Offens, Loes Teeuwen, Gozde Gucluler Akpinar, Loïc Steiner, Sander Kooijmans, Doste Mamand, Hannah Weissinger, Alexander Käll, Maria Eldh, Oscar P.B Wiklander, Samir EL Andaloussi, Mikael C.I. Karlsson, Pieter Vader, Susanne Gabrielsson. A fusion protein that targets antigen-loaded extracellular vesicles to B cells enhances antigen-specific T cell expansion. [Manuscript]

III. Extracellular vesicles from bovine milk and serum can be loaded with protein antigen by lyophilization and induce CD4+ T cell responses in mice. Annemarijn Offens, Maria Eldh, Loes Teeuwen, Gozde Gucluler Akpinar, Loïc Steiner, Rebeca Cardoso, Monisha Samuel, Eduardo Villablanca, Susanne Gabrielsson. [Manuscript]

IV. Loïc Steiner, Maria Eldh, Annemarijn Offens, Rosanne E. Veerman, Markus Johansson, Tammer Hemdan, Hans Netterling, Ylva Huge, Abdul-Sattar Aljabery Firas, Farhood Alamdari, Oskar Liden, Amir Sherif and Susanne Gabrielsson. Protein profile in urinary extracellular vesicles is a marker of malignancy and correlates with muscle invasiveness in urinary bladder cancer. Cancer Letters. 609, 217352 (2025). https://doi.org/10.1016/j.canlet.2024.217352