DESIGN OF HYALURONIC ACID-STING CONJUGATE TO ENHANCE THE LOCAL IMMUNE RESPONSE OF GLIOBLASTOMA

Details

Supervisors

Préat, Véronique

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Glioblastoma (GBM) is the most aggressive brain tumor with a median survival of approximately 15 months. GBM patients are normally treated by surgery to remove the main tumor mass, followed by radiation and temozolomide chemotherapy to target residual tumor cells that can induce recurrence. The failure of therapies could be addressed by the presence of the blood-brain barrier and high chemoresistance rendering it an unmet clinical need. Immunotherapy has gained attention for the treatment of several cancers, including GBM. Unfortunately, the human GBM is a cold tumor with extremely low infiltration of T cells and an immunosuppressive tumor microenvironment (TME) hampering the success of immunotherapeutic agents. Recently, the STimulator of the INterferon Genes (STING) pathway has been a potential target for cancer treatment which activates the immune response via the production of type I interferons and immune-stimulatory cytokines. While STING agonists have been extensively studied, their potential role in regulating the immune response against GBM has not yet been investigated. In this study, we hypothesized that the conjugation of a STING agonist (MSA-2) to Hyaluronic Acid (HA) could enhance the performance of the free drug after in situ administration, eliciting a potent immune response against GBM overcoming the immunosuppressive TME. HA was selected for its biocompatibility and high affinity toward the CD44 receptor, which is highly expressed in GBM and on immune-cell membranes. First, we set up a synthesis procedure to conjugate MSA-2 to HA. The novel conjugate was characterized via 1H NMR, UV-Vis, size and zeta potential showing the chemical identity of the new conjugate. Next, we evaluated the ability of HA-MSA2 to activate STING pathway in different cell lines (BV-2, J774A.1, JAWS II) by quantitative real-time PCR (qRT-PCR) analysis of expression levels of IFNβ, and other antitumor effectors of STING activation, namely TNFα, IL6 and CXCL10. Results showed that HA-MSA2 induced a prolonged and sustained expression of these effectors in the analysed cell lines compared to the free drug. This was also investigated in two different GBM cell lines (SB28 and GL261) as the STING pathway is also expressed in cancer cells. The results showed that there is a significant production of IFNβ, IL6, TNFα and CXCL10 even at 24 h. Moreover, we observed that HA-MSA2 conjugate decreased the viability of the SB28 cell line. Owing to their high efficiency cytosolic drug delivery capacity, the median inhibitory concentration (IC50) dose of HA-MSA2 was at least 10-fold lower than the IC50 dose of unconjugated MSA-2. Based on previous works reported in the literature, we hypothesized that HA-MSA2 could induce immunogenic cell death (ICD) by triggering the expression of danger signals like adenosine triphosphate (ATP) release and the translocation of calreticulin on the cancer cell membrane. We observed that the ATP release in SB28 cells spiked by one thousand times compared to the control. These results are consistent with the in vitro maturation of the different cell lines mediated by incubation of conditioned medium from SB28 glioma cells treated with HA-MSA2: the percentage of CD86+ cells doubled suggesting the activation of these cells by the conjugate. Furthermore, the effects of the in vivo administration of HA-MSA2 on the immunosuppressive GBM TME, tumor growth, and median survival were evaluated in C57BL/6J SB28 tumor-bearing mice model. The mice were treated intratumorally after 10 days of grafting with the HA-MSA2 together with an anti-PD1 antibody injected intraperitoneally. Interestingly, the median survival was prolonged by 6 days after the administration of our conjugate. The brain samples were collected and analysed at the endpoint by flow cytometry for the different immune markers. HA-MSA2 generated a significantly higher percentage of innate immunity markers like DCs and NK cells. An increment of CD8+ T cells was observed suggesting that the conjugate also contributed to adaptive immunity. Finally, the results highlight a decrease in granulocytic-derived neutrophils and a higher CD8+ T cells/Tregs ratio, suggesting that HA-MSA2 could re-educate the GBM TME.