Poster X14 - Optimizing the Simultaneous Delivery of a VISTA Checkpoint Inhibitor and TLR Agonist

Introduction: Immune checkpoint inhibitors are powerful tools in cancer immunotherapy, eliminating a key mechanism in tumor immune evasion. V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel immune checkpoint highly expressed across myeloid and T cell populations within the immunosuppressive tumor microenvironment. It has also been shown to inhibit TLR signaling, a pro-inflammatory pathway in antigen-presenting cells activated by pathogen-associated molecules. Knockout of VISTA enhanced their ability to produce pro-inflammatory cytokines [1]. In previous work, we designed a “dual” lipid nanoparticle (LNP) containing a VISTA siRNA and a TLR9 agonist (CpG) to couple this synergy with the advantages of a nanoparticle delivery system. Lipid nanoparticles have been popular vehicles for nucleic acid delivery due to their ability to shield their cargo from enzymatic degradation and facilitate cellular uptake. Furthermore, the dual LNP ensures that both cargos are simultaneously delivered to the same antigen-presenting cell. It was shown in several mouse tumor models that the dual LNP induced a potent anti-tumor response and improved immune cell infiltration into the tumor.

In this work, we perform mechanistic studies to explore the role of each component, the relationship between them, and their dosages in the dual LNP. A major advantage of nanoparticle delivery is its customizability, especially in combination therapies. One has much more control over the ratio of therapeutics and dosage each cell experiences. We then optimize the previous dual LNP by applying these new parameters and compare the performance between the two formulations.

Materials and

Methods: Lipid nanoparticles were made with DLin-MC3, DSPC, cholesterol, DSPE-PEG, and DMG-PEG. Lipids were suspended in ethanol, and nucleic acids were suspended in acetate buffer (pH 4). The two components were combined at a 1:3 ratio, sonicated, and left to dialyze in PBS overnight. Lipid nanoparticles were characterized through size analysis (DLS) and loading efficiency (RNA/DNA quantification assay).

The ratio of VISTA siRNA to CpG and the N/P ratio were both explored in these studies. The composition of the tested dual LNPs ranged from 0% CpG/100% VISTA siRNA to 100% CpG/0% VISTA siRNA. The N/P ratio is the ratio between cationic lipid (DLin-MC3) and nucleic acid cargo and can be inversely correlated to the dose per nanoparticle. N/P ratios of 3, 6, and 9 were tested in this work. In addition, control dual LNPs were made with GpC and non-targeting siRNA.

Cell culture experiments were conducted with RAW264.7 murine macrophages and DC2. 4 murine dendritic cells. To study cytokine release, cells were treated with 3 μg/mL of CpG, and cell culture supernatant was collected 6 and 24 hours after treatment. Cytokine concentrations were analyzed with ELISAs. To study protein expression, cells were treated with 50 nM of VISTA siRNA and harvested for flow cytometry 24, 36, and 48 hours after treatment. VISTA and CD80 expression were evaluated through the mean fluorescent intensity (MFI) of their respective fluorophores.

Results, Conclusions, and Discussions: Results and Discussion
The gene silencing and immunostimulatory performance of the dual LNP were both affected by altering the ratio of VISTA siRNA to CpG and changing the total dose of nucleic acid per nanoparticle. A 100% CpG dual LNP nearly doubled VISTA expression at 24 hours (Figure 1). This upregulation is not unexpected, as the expression of immune checkpoint proteins typically increases in response to inflammatory signals. Interestingly, the dual LNP with 28% CpG outperformed the 0% CpG formulation, suggesting that CpG may also contribute to VISTA silencing. Similarly, a dual LNP formulation with 52% CpG induced the highest TNFα secretion (Figure 2). This bell-shaped trend indicates that cytokine release is not linearly dependent on the proportion of CpG in the dual LNP, and there may be a minimum level of VISTA silencing required to enhance TLR signaling. A relationship between dose per nanoparticle and dual LNP performance was also established. Finally, an optimized dual formulation was developed by combining the optimal conditions from the two previous studies.

Conclusions
The co-delivery of VISTA siRNA and CpG within the dual LNP is a potent combination therapy that can simultaneously silence an immune checkpoint and provide an inflammatory stimulus to the tumor microenvironment. However, the two molecules have an intricate biological relationship. Through optimizing the dose within the nanoparticle, we redesigned the dual LNP to maximize the performance of the individual components while enhancing their synergy. This was evident in both VISTA silencing and cytokine secretion data. Moving forward, we plan to evaluate the optimized dual LNP in vivo to demonstrate that these changes translate to a more robust anti-tumor response.

Acknowledgements (Optional): This work was supported by grants from the National Cancer Institute R01CA253627 (E.K.), R01CA278633 (E.K. ), P30CA043703 (E.K.), and the Angie Fowler AYA Cancer Research Fund (E.K.). T.J.M. was supported by the NSF graduate research fellowships program.

References
1. Xu, W., Dong, J., Zheng, Y., Zhou, J., Yuan, Y., Ta, H. M., Miller, H. E., Olson, M., Rajasekaran, K., Ernstoff, M. S., Wang, D., Malarkannan, S., & Wang, L. (2019). Immune-Checkpoint Protein VISTA Regulates Antitumor Immunity by Controlling Myeloid Cell-Mediated Inflammation and Immunosuppression. Cancer immunology research, 7(9), 1497–1510. https://doi.org/10.1158/2326-6066.CIR-18-0489