Local Reprogramming Nanoparticles Generate Systemic Immunity and Potent Anti-Tumor Responses in Vivo

Introduction: Novel therapeutic strategies that can generate safe, targeted, widespread anti-cancer immunity are needed, particularly for cases that do not respond to immune checkpoint inhibitors (ICIs) and where metastatic disease is present. Systemic cytokine therapies are often associated with high toxicities; a locally administered therapeutic capable of safely generating systemic immune effects would be highly advantageous in addressing cancers that extend beyond a primary tumor. Here, we use a metastatic model of melanoma to evaluate whether locally administered reprogramming nanoparticles can sensitize resistant cancer to ICI and elicit potent anti-tumor responses, at both local and distant sites.

Materials and

Methods: B16F10 tumors were established on the flanks of C57BL/6J mice. On day 8, B16F10 cells engineered to stably express luciferase (B16F10-fLuc) were administered via the tail vein. On days 9, 11, 16, and 18, poly(beta-amino ester)-based NPs delivering a control plasmid (GFP) or co-delivering plasmids encoding 4-1BBL and IL-12 were administered to flank tumors, and 100 µg anti-PD1 was administered intraperitoneally. On day 19, flank tumors were measured, lung tumor burden was visualized via In Vivo Imaging System (IVIS), then mice were sacrificed and lungs and flank tumors were harvested. A portion of each lung and flank tumor was fixed for sectioning and immunofluorescent (IF) staining for Sox10 and CD8. Stained slides were analyzed using HALO. Statistical analysis was performed in GraphPad Prism. Significance assessed via one-way ANOVA with Tukey’s multiple comparisons ( **, p< 0.01; ***, p< 0.001; ****, p< 0.0001).

Results, Conclusions, and Discussions: In mice with primary B16F10 flank tumors and metastases in the lungs, locally administered control NPs with systemic anti-PD1 did not control lung tumor burden (Figure 1, top). When 4-1BBL/IL-12 NPs were locally administered with systemic anti-PD1, however, tumor burden was visibly reduced (Figure 1, bottom). IF staining of lung sections revealed uninflamed metastatic deposits of Sox10+ melanoma cells in the lungs of mice receiving control NPs and anti-PD1 (Figure 2A, left), whereas lungs of mice that received 4-1BBL/IL-12 NPs and anti-PD1 showed CD8+ T-cell infiltration and minimal Sox10+ melanoma (Figure 2A, middle), more closely resembling naïve lungs (Figure 2A, right). IF staining of matched flank tumors revealed a highly inflamed tumor microenvironment following treatment with 4-1BBL/IL-12 NPs and anti-PD1 (Figure 2B). Image analysis and quantification of Sox10+ cells showed that 4-1BBL/IL-12 NPs with anti-PD1 led to significantly fewer melanoma cells in the lungs compared to control (Figure 3A, left), and this trend was confirmed via quantification of bioluminescent tumor cells visualized via IVIS (Figure 3A, right). Most notably, treatment with 4-1BBL/IL-12 NPs in the flank tumor and systemic anti-PD1 reduced lung melanoma burden to the extent that it was not statistically different from naïve lungs via either measurement (Figure 3A-B). Lung tumor burden, measured via IVIS, correlated negatively with CD8+ T-cell density in matched flank tumors (Figure 4A, R2 = 0.631, p = 0.0001) and also correlated with flank tumor growth (Figure 4B, R2 = 0.863, p < 0.0001). Together, these results suggest that local immune priming is required for a systemic response, and cases with strong local responses also result in the greatest systemic responses. Overall, this demonstrates the utility of locally injected reprogramming nanoparticles to sensitize a resistant model to ICI, leading to productive local and systemic immune responses.

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