Advancing Cancer Immunotherapy with Exosomes

Genetically Engineered Exosomes Elicit Targeted Anti-Cancer Immunity

What is Immunotherapy?

Cancer immunotherapy is a type of cancer treatment that utilizes the patient’s own immune system to fight cancer cells, either by stimulating the immune system’s ability to target and attack cancer cells or by using synthetic substances that behave like naturally occurring immune system components¹. Traditional cancer treatments, like chemotherapy, are only effective when the drugs are present in the body and impact healthy tissues as well as cancer cells, which results in extreme side effects, such as hair loss, anemia, nausea, and increased likelihood of infection. The objective of cancer immunotherapy is to provide a long-term cancer-fighting response that overcomes these adverse side effects by using the immune system to directly target cancer cells. Furthermore, since immunotherapy relies on the immune system’s ability to target cancer cells, it can potentially be effective against any type of cancer.

A Novel Solution Using Multifunctional Exosomes

Exosomes are naturally occurring nanovesicles recognized for their therapeutic capability, primarily as vehicles for drug delivery. Recently, there has been significant interest in using exosomes for cancer immunotherapy. In a study published in Molecular Therapy, cancer researchers with the University of Southern California explored the potential of expressing both antibody targeting groups and immunomodulatory proteins on the surface of exosomes for cancer immunotherapy². The authors reasoned that these multifunctional exosomes might be able to elicit a better immune response and thus improve therapeutic efficacy because unlike current approaches to immunotherapy, one exosome could both guide and stimulate T cells towards killing cancer cells. They tested their hypothesis by designing a genetically engineered multifunctional immune-modulating exosome (GEMINI-Exos) with monoclonal antibodies specific for human T cells and cancer cells as well as immune checkpoint modulators fused to exosomal membrane proteins. The effectiveness of GEMINI-Exos was then evaluated in cellular and animal models of triple-negative breast cancer (TNBC).

Creation of GEMINI-Exos

Four proteins were selected for expression in the engineered exosome to generate anti-cancer immunity: αCD3, αEGFR, OX40 Ligand (OX40L), and programmed death 1 (PD-1). CD3 is a T-cell co-receptor and EGFR is a growth factor receptor commonly overexpressed in cancer cells³. The surface-displayed monoclonal antibodies (αCD3, αEGFR) expressed by GEMINI-Exos direct T-cells to EGFR-positive TNBC tumors. OX40L binds to the T-cell receptor OX40, an immune checkpoint modulator, to help stimulate an immune response⁴. Conversely, when the T-cell receptor PD-1 is bound by its ligands (PD-L1/L2), it produces an inhibitory signal⁵. Cancer cells will often express PD-L1/L2 ligands to inactivate T cells and evade detection by the immune system. GEMINI-Exos-expressed PD-1 binds the PD-L1/L2 ligands, effectively blocking cancer cells from inactivating T cells.

Fusion gene fragments for the proteins were created by overlap extension PCR and integrated into a plasmid mammalian expression vector using restriction enzyme cloning. Plasmid vectors were purified using ZymoPURE II Plasmid Kits to produce transfection-grade, ultra-pure plasmid DNA. GEMINI-Exos were generated by transiently transfecting the purified fusion gene constructs into Expi293F cells, which is a human cell line optimized for transient protein expression. Centrifugation was used to isolate the genetically modified exosomes from cell culture supernatant for downstream analysis.

Promising Results for Versatile, Specific Immunotherapy

Immunoblot data revealed expression of the fusion proteins in the exosomes and ELISA and flow cytometry results confirmed strong binding affinity of the engineered exosomes to their protein targets and cells. Furthermore, mice treated with GEMINI-Exos exhibited the greatest tumor growth inhibition compared to combined or separate treatment with exosomes displaying only αCD3 and αEGFR or OX40L and PD-1. Tumors from GEMINI-Exos treated mice also contained the highest percentage of CD8+ T cells and ratio of CD8+T cells to regulatory T cells. This suggests that co-expression of both surface-displayed antibody targeting groups and immunomodulatory proteins on the same exosome vesicle is a more effective treatment than expressing them on separate exosomes. In addition, no overt toxicity or significant weight loss was observed in the treated mice.

These promising results and the potential adaptability of this technique to other kinds of cancers demonstrate the potential of genetically modified multifunctional exosomes as a potent, safe, and versatile method for cancer immunotherapy.

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References

1. American Cancer Society. How Immunotherapy is Used to Treat Cancer. Retrieved June 17th, 2024, from https://www.cancer.org/cancer/managing-cancer/treatment-types/immunotherapy/what-is-immunotherapy.html
2. Cheng, Q., Dai, Z., Smbatyan, G., Epstein, A. L., Lenz, H., & Zhang, Y. (2022) Eliciting anti-cancer immunity by genetically engineered multifunctional exosomes. Molecular Therapy, 30(9), 3066 – 3077.
3. Wieduwilt, M. J., Moasser, M. M. (2008) The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci., 65(10), 1566 – 1584.
4. Ishii, N., Takahashi, T., Soroosh, P., Sugamura, K. (2010) OX40-OX40 ligand interaction in T-cell-mediated immunity and immunopathology. Adv. Immunol., 105, 63 – 98.
5. Jin, H. T., Ahmed, R., Okazaki, T. (2010) Role of PD-1 in Regulating T-Cell Immunity. In: Ahmed, R., Honjo, T. (eds) Negative Co-Receptors and Ligands. Current Topics in Microbiology and Immunology, 350, 17 – 37.