Targeting tumor-derived exosomes using a lectin affinity hemofiltration device
Annette M Marleau1, Michael T Jacobs1, Nadia Gruber1, Timothy C Rodell1, Soldano Ferrone2, Theresa L Whiteside3
1Aethlon Medical, Inc., San Diego, CA, United States
2Massachusetts General Hospital, Boston, MA, United States
3University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
Background: Tumor-derived exosomes (TEX) are 30-150 nm vesicles that are released by tumor cells and have systemic disease-promoting effects. Despite abundant research showing that TEX are purveyors of tumor growth-promoting and immunosuppressive cargo, a clinical strategy for addressing exosomes in oncology is lacking. Aethlon Medical is initiating clinical trials in cancer patients with the Hemopurifier®, a hollow fiber hemofiltration device that is operated using dialysis infrastructure, for removing exosomes from circulating blood. The Hemopurifier incorporates size exclusion and a lectin (Galanthus nivalis agglutinin; GNA) affinity matrix for capturing nanoparticles having high mannose glycoprotein surface structures.

Experimental Procedures: A benchtop version of the Hemopurifier was operated by recirculating samples of cancer patients’ plasma through the hollow fiber filters ex vivo. The exosomes remaining in plasma were quantified using nanoparticle tracking analysis measurements and the ExoView instrument. To identify the Hemopurifier-bound material, an elution technique was developed to displace the GNA-bound material from the Hemopurifier. The recovered vesicles were assessed using ExoView and flow cytometry and compared to an established exosome isolation method, size exclusion chromatography, which utilizes small-size ion exchange columns (mini-SEC). In vitro assays were also performed by co-culturing the recovered exosomes with T cells to evaluate their immunosuppressive functions.

Results: The results demonstrate that the GNA affinity capture mechanism of the Hemopurifier is effective for clearing 92-99% of exosomes from input concentrations of 109-1010 exosomes per mL of plasma. The device captures exosomes from diverse tumor types including head and neck cancer, melanoma, ovarian cancer, esophageal cancer and breast cancer. Analysis of plasma samples from Stages III and IV triple negative and human epidermal growth factor receptor 2 overexpressing breast cancer showed that Hemopurifier-bound material comprised the appropriately-sized vesicles expressing tumor susceptibility gene 101 and tetraspanins. In comparison to mini-SEC-isolated exosomes, tumor-specific epitopes and immunosuppressive proteins exhibited equivalent expression among Hemopurifier-isolated exosomes. The immunosuppressive functions of Hemopurifier- and mini-SEC-isolated exosomes were also equivalent.

Conclusions: The Hemopurifier effectively clears exosomes present in plasma that originate from diverse types of cancer. The Hemopurifier-captured exosomes exhibit signatures of malignancy and immunosuppression and are therefore relevant therapeutic targets.

This research was supported by the National Cancer Institute of the National Institutes of Health under award number 1R43CA232977-01.

Session: Immunomodulatory Agents and Interventions 1 (Virtual Poster Session)
Category: IMMUNOLOGY: Preclinical and Clinical