Development of a Dual-Action Lipid Nanoparticle for Gene Therapy Checkpoint Blockade Immunotherapy Treatment of Solid Tumors

Introduction: The root of cancer lies within genetic mutations that leads to uncontrollable cell proliferation. Gene therapies hold significant promise for stably or transiently introducing proteins that are lacking in tumor cells or silencing upregulated pro-tumoral proteins. Gene silencing via siRNA provides an alternative approach to checkpoint blockade immunotherapy, which is typically executed via antibody therapy. The main advantage of gene silencing is more durable knockout of the checkpoint protein with fewer treatments. Delivery platforms are developed for encapsulation, protection, and delivery of nucleotide-based molecules. Nanoparticle delivery of cargo mediates preferential delivery to immune cells in the tumor microenvironment over cancer cells themselves. This is advantageous when the cargo is immune stimulants or checkpoint blockades that specifically affect phagocytic myeloid immune cells.
We investigate the use of lipid nanoparticle delivery platforms for delivery of gene therapy to mediate VISTA (V-Domain Immunoglobulin Suppressor of T Cell Activation) immune checkpoint blockade. VISTA is found upregulated on myeloid and T cells in solid tumor microenvironments (TME) and shuts down anti-tumor T cell responses when interacting with its ligand. Studies have shown that VISTA inhibition sensitizes innate immune cells to pro-inflammatory toll-like receptor (TLR)-mediated activation. Based on this concept, we designed a Dual-LNP to co-deliver a VISTA-targeting siRNA and the TLR9 agonist CPG dinucleotide to synergistically elicit robust anti-tumor responses and clearance in solid tumor models. Considering that nanoparticles are preferentially phagocytosed by tumor-associated innate immune cells, we hypothesized that the simultaneous silencing of VISTA and stimulation of TLR9 will generate potent antitumor immune responses.

Materials and

Methods: Lipid nanoparticle formulations were made with D-Lin-MC3 ionizable cationic lipid DSPC, cholesterol, DMG-PEG2000, and DSPE-mPEG2000 at a molar ratio of 50:10.5:38:1.4:0.1. VISTA siRNA and CPG ODN-1826 were resuspended in nuclease free acetate buffer, pH4. Lipids in ethanol were rapidly mixed with VISTA siRNA and/or CPG ODN-1826 at a ratio of 1:3 (v/v). The solution was then probe sonicated and dialyzed against nuclease free PBS. Fluorescently tagged nanoparticles were made the same way, with the addition of up to 0.5 mol% DiR’. Equivalent molar percentage was taken away from cholesterol. Nanoparticles were analyzed for hydrodynamic size using DLS and zeta potential in PBS. Drug loading efficiency and encapsulation efficiency were determined using Quant-it Ribo-green or Oli-green kits.
Biodistribution of Dual-LNPs were investigated following single Dual-LNP treatment of orthotopic YUMM1.7 tumor bearing mice. Mice were intratumorally injected with DiR’ tagged Dual-LNPs equivalent to 4ug CPG and 10ug VISTA siRNA, 8 days following inoculation. 24 hours later, tumor, liver, spleen, or tumor draining lymph node were harvest. Organs were processed into single cell suspensions. Cells were analyzed for cellular uptake of nanoparticle and phenotype via flow cytometry (BDFortessa).
Survival studies investigated orthotopic YUMM1.7 and B16F10 tumor bearing mice and flank MC38 tumor bearing mice. Mice were treated once tumors reached ~70mm3. Mice received intratumorally Dual treatments every 7 days for 4 rounds of treatment. After every dual treatment, mice received additional siRNA LNP treatment. Rechallenge studies were performed by flank injection of tumor cells in opposite flank to original inoculation.

Results, Conclusions, and Discussions: This study reports the development of a dual action lipid nanoparticle (Dual LNP) that consistently and efficiently incorporates VISTA-targeting siRNA and TLR9 agonist CpG oligonucleotide. Our results show that the Dual LNP ensured co-delivery of its synergistic cargoes to tumor-infiltrating myeloid cells, leading to simultaneous silencing of VISTA and stimulation of TLR9. Further, local administration of Dual LNP offered significant advantages including direct antitumor immune response within the primary tumor and reduced systemic immune related adverse events. The lack of the Dual LNP “spill” into the blood circulation and sequestration of therapy in the TME enhanced therapeutic efficacy while minimizing potential systemic toxicity. Following local injection of the Dual LNPs, over 80% of the LNP+ cells were CD45+. Additionally, around 74% of the LNP+ cells were myeloid immune cells. Of the myeloid immune cell subset, 90% of MDSCs, 70% of tumor associated macrophages, and 57% of DCs were Dual-LNP+. Thus, the Dual LNP ensures the uptake of both cargoes by the same APCs in the tumor.
Mechanistically, the Dual LNP leverages the simultaneous immune activation by TLR9 stimulation with silencing the gene expression of VISTA. We show that the Dual LNP synergistically augmented antitumor immune responses and rejected large established tumors whereas LNPs containing VISTA siRNA or CpG dinucleotides alone were ineffective. The Dual LNP treatment achieved an 83% cure rate in MC38 colon carcinoma, 60% B16F10 melanoma, and 47% cure rate in YUMM1.7 melanoma tumors. Further, cured mice exhibit immunological memory and reject tumor recurrence upon reinjection of cancer cells, without the need for additional therapeutic intervention. In comparison with therapies using the soluble CpG and a VISTA-specific monoclonal antibody which failed to control tumor growth or yield any cure. This further demonstrates that the gene therapy-mediated immunomodulation is significantly more effective compared to immune checkpoint blockade using antibodies.

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. A.B. was supported by a fellowship from the NIH Interdisciplinary Biomedical Imaging Training Program (T32EB007509).