Poster CC11 - Effective immunostimulatory action of Dual Action Nanoparticle in innate and adaptive immune cells in a Mouse Melanoma Model

Introduction: Cancer immunotherapy aims to re-engage pro-inflammatory immune cells to drive robust anti-tumor responses. However, tumor microenvironments exist in a massively immunosuppressed state, which poses a major challenge for the success of many cancer immunotherapies. Upregulation of immune checkpoint proteins severely limits the capacity of cytotoxic response by tumor resident immune cells. Mechanistic studies have identified the role of checkpoint proteins like PD-L1, PD-1, CTLA-4. Targeting and blocking this B7 family of immune checkpoint receptors via monoclonal antibodies has yielded durable clinical outcomes. However, recurrent and metastatic disease remains prevalent in patients following resistance to checkpoint blockade therapy.
To overcome these limitations, a two-pronged attack was designed to 1) stimulate the pro-inflammatory immune response of myeloid cells and 2) silence an immune checkpoint on tumor-associated myeloid immune cells. We chose to downregulate a novel checkpoint protein VISTA (V-domain Immunoglobulin Suppressor of T cell Activation). When activated, VISTA impairs toll-like receptor (TLR) pro-inflammatory activity, promotes myeloid derived suppressor cell expansion, and shuts down anti-tumor T cell function. Eliminating the VISTA checkpoint sensitizes immune cells to TLR stimulation and allows for potent cytotoxic T cell activity. We downregulated VISTA via siRNA gene therapy, co-delivered with CPG TLR9 agonist, via a dual action lipid nanoparticle (Dual-LNP). Local administration of Dual-LNP therapy has yielded significant cure in multiple tumor models. We aimed to elucidate the mechanism of action of the Dual-LNP therapy and global changes in the tumor immune microenvironment. Specifically, we investigated the phenotypic changes in the myeloid and T cells during Dual-LNP treatment.

Materials and

Methods: C57BL6/J mice were orthotopic inoculating with YUMM1.7 tumors. Mice were treated once tumors reached ~70mm3. Mice received intratumorally Dual treatments every 7 days for 1-2 rounds of treatment. After every dual treatment, mice received additional siRNA LNP treatment. 5 days of 12 days after the start of treatment, tumors and tumor draining lymph nodes were harvest. Organs were processed into single cells suspensions and then stained with fluorescent antibodies to identify macrophages, dendritic cells, MDSCs, and T cells with additional markers like VISTA expression, MHCII expansion, and T cell activation markers.
Tumors analyzed for cytokine secretion were homogenized in PBS. Supernatant was analyzed with ELISA analysis. Tumors analyzed for T cell activatability were harvested 12 days after the start of treatment and processed into single cell suspensions under sterile conditions. The final cell pellet was resuspended in complete, sterile RPMI media. A portion of the cell suspension was plated in sterile U-bottom 96 well plates (2 replicates per mouse). anti-CD3 (clone 2C11, Biolegend) and anti-CD28 (clone PV1, BioXCell) were added to each of the samples for final concentrations of 2.5 μg/mL 2C11 and 1ug/mL PV-1. Cells were incubated at 37°C, 5% CO2 for 15 hours. 1x Monesin (Biolegend) and 1x Brefeldin A (Biolegend) were added and then incubated for an additional 4.5 hours before staining for surface proteins and intracellular cytokines.

Results, Conclusions, and Discussions: Using the YUMM1.7 murine melanoma model, which possesses similar mutations to human melanomas, we show that the Dual LNP synergistically enhanced cytotoxic immune responses in solid melanoma tumors leading to a 47% cure rate and protection against recurrence. Tumor rejection was associated with influx of immune cells to tumor tissues, production of proinflammatory cytokines, and improved function of cytotoxic T cells. By simultaneous delivering an immunostimulatory cargo and a checkpoint blockade siRNA, the Dual-LNP therapy facilitated reprogramming of tumor-associated myeloid cells, enhancement of the effector function of CTLs, and promotion of memory T cell responses.
The goal of the two-pronged attack aimed at silencing the VISTA checkpoint while also activating the same myeloid immune cell in the TME. The Dual-LNP successfully achieved VISTA expression downregulation in macrophages and dendritic cells, while also showing expanded MHCII+ expressing proportions of each population, indicating antigen presentation. There was an almost complete elimination of immunosuppressive M-MDSCs (from ~12% to 0.5%). The reduction in M-MDSCs could stem from differentiation into inflammatory DCs as supported by the 7.5x increase in the percentage of Ly6C+ DCs. Further, the cytokine profile in the TME was remarkably more pro-inflammatory, with TNF-α, IFN-γ, IL-12p40, and IL-2 all upregulated. This shows that as the Dual-LNP was able to decrease VISTA expression and simultaneously induce robust TLR mediated pro-inflammatory cytokine secretion and APC activity in the local TME.
Thus, we would expect enhanced T cell priming activity and investigated the tumor draining lymph node and found a 2x increase in myeloid cell presence in tumor draining lymph nodes following Dual-LNP treatment, including antigen presenting macrophages. Mechanistically, this shows that the Dual-LNP treatment restored TLR mediated APC activity towards T cell priming. Efficient T cell priming and restoration of a pro-inflammatory TME led to greater activatability of tumor resident T cells, as seen in the larger proportion of Granzyme B+ and IFN-γ+ cytotoxic CD8+ T cells and subsequent robust tumor clearance. Following full tumor clearance, treated mice also had more tumor specific reactive T cells found systemically, which prevents long term recurrence of the cancer.

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).