Poster CC18 - Glioblastoma Immunotherapy using RIG-I Activating Nanoparticles

Introduction: Glioblastoma (GBM) is a rare form of brain cancer with over 13,000 new cases each year and a dismal outlook for patients who face a mean survival time of 12-18 months post-diagnosis. Further, there is a high recurrence rate with a five-year survival rate below 10%. The current standard of care – comprising surgical resection followed by radiation and chemotherapy – has not advanced significantly over the past 20 years, creating a need for new therapies. To this end, leveraging pattern recognition receptor (PRR) activation to elicit strong innate immune responses shows promise for cancer immunotherapy. RIG-I is one such PRR, and higher levels of RIG-I are associated with improved survival outcomes for the first year following GBM diagnosis. RIG-I recognizes short double-stranded RNA with a triphosphate (3p) group on the 5’ end (3pRNA), and RIG-I activation stimulates pro-inflammatory responses, including type-I interferons which trigger antitumor immunity. At the preclinical level, RIG-I activating therapies have shown promise for treating various solid tumors, yet pharmacological activation of RIG-I has not yet been explored for GBM immunotherapy. Despite their promise for cancer immunotherapy, the clinical utility of 3pRNA RIG-I agonists is currently limited by significant drug delivery barriers, including poor intracellular uptake, nuclease degradation in the endosome/lysosome, and an inability to reach the cytosol to bind RIG-I. To realize the promise of RIG-I, we will use RIG-I activating nanoparticles (RANs) to treat a mouse model of glioblastoma. RANs are pH-responsive and will deliver a novel 44-base pair stem-loop RNA (SLR20).

Materials and

Methods: Di-block polymer consisted of a first block of 10 kDa methoxy-polyethylene glycol (mPEG) and a second block of Pyridyl disulfide ethyl methacrylate poly[(DMAEMA-c-butyl methacrylate (BMA)) (PDB). The DB block serves to complex with RNA. To form micelles, the di-block copolymer was dissolved in ethanol at 100 mg/mL then diluted with sterile-filtered HEPES-buffered glucose solution to a concentration of 10 mg/mL. RNA was complexed to NPs for 45 minutes at room temperature, then Ribogreen quantified RNA loading and dynamic light scatter (DLS) quantified size. The activity of RANs was assessed using Quanti-luc and Quanti-blue assays in THP1-Dual (human monocytes) and A549-Dual (human lung carcinoma) reporter cell lines. Mouse GL261 or CT2A cells were treated with RANs, then 24 hours later, supernatants were collected for ELISA, and cells were stained for flow cytometry. The ELISA was conducted according to the manufacturer’s protocol, and flow cytometry used a CellStream® Flow Cytometer. In vivo Experiments: Mice were inoculated with 1,000,000 GL261 or CT2A cells on the right flank. When tumors reached ~50 mm3, mice were treated intratumorally with PBS or 10 µg of RANs, with additional injections on days 3 and 6 post treatment initiation. Orthotopic tumors were engrafted using 200,000 CT2A-luciferase cells at coordinates of 1.00 mm anterior, 2.00 mm lateral, and 3.00 mm deep using a 25G Hamilton syringe. Mice were treated intratumorally with 2.5, 5, or 10 µg of RANs 11 days later using the same injection coordinates as engraftment. Tumor volume was quantified every other day using IVIS.

Results, Conclusions, and Discussions: In vitro experiments: DLS showed that RANs were ~90 nm in diameter, and Ribogreen showed successful complexation of RNA to nanoparticles. RANs also activate antigen presenting cells, with A549-Dual (Fig. 1A) and THP1-Dual (Fig. 1B) producing more type one IFN in response to RAN treatment compared to control RANs (cRANs) with inactive RNA. Additionally, we show that GL261 and CT2A cells treated in vitro with RANs produced significantly more interferon beta (Fig. 1C-D) and had increased MHC I expression following RAN treatment (Fig. 1E-F) compared to cells treated with cRANs. Interestingly, the more immunogenic GL261 cells had differences in frequency and MFI of MHC I, while CT2A cells only had differences in frequency. This suggests that RANs more potently activate GL261 cells because more cells express MHC I, and the cells that express it have elevated levels.
In vivo experiments: RANs significantly delayed tumor growth and prolonged survival in both GL261 and CT2A flank tumors compared to cRANs and vehicle treated mice (Fig. 1G-H). Specifically, RAN treatment extended the time to first death by 6 days, and 60% of mice are long-term survivors with tumor volumes below 500 mm3. While the response to RANs was not as pronounced in CT2A tumors, there were survival benefits compared to cRANs which had similar survival to vehicle treated controls. Initial dose finding studies in the orthotopic GBM model found that a 5 µg dose of RANs is the maximum tolerated dose, as mice treated with higher doses experience substantial amounts of weight loss. Additionally, preliminary results suggest that the 5 µg dose of RANs can delay tumor growth, but it will require further studies to see if these changes are significant compared to cRANs (Fig. 1I). Herein, we show that RANs can effectively activate antigen presenting cells and cancer cells in vitro, delay growth of flank tumors, and find a maximum tolerated dose in the brain. Overall, RANs display preclinical promise to combat a deadly cancer.

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