Poster O4 - Modular Polymeric Nanoparticles Enable Systematic Evaluation of Combinatorial Immunostimulatory Gene Delivery to Melanoma Tumors

Introduction: T-cell activation relies on signal 1 (peptide presented via MHC to TCR), signal 2 (co-stimulation), and signal 3 (secreted cytokine) for productive immune responses. Checkpoint blockade has become the standard of care for a variety of solid tumors, though many tumors are resistant to immune checkpoint inhibitors, and novel therapeutics to address cancer are needed. Non-viral gene delivery has shown immense promise in modulating the tumor microenvironment leading to anti-cancer immune responses in vitro and in vivo. Here, we use a modular polymeric nanoparticle delivery system as a tool to investigate co-delivery of plasmids encoding various signal 2 and signal 3 molecules for local immune activation against B16F10 melanoma and profile outcomes in vivo.

Materials and

Methods: B16F10 tumors were implanted in the flanks of C57BL/6J mice. On days 9, 11, 16, and 18, mice received intratumoral injections of poly(beta-amino ester)-based NPs delivering a control plasmid (luciferase) or combinations of plasmids encoding a signal 3 molecule (IL-2, IL-12, IL-15, IL-23, IL-35, GM-CSF) and a signal 2 molecule (4-1BBL, CD80, CD86, OX40L). Tumors were measured and harvested on day 20 for analysis via intracellular cytokine staining and flow cytometry. To assess survival, NPs were administered to B16F10 tumors on days 9, 11, 16, and 18, and tumor size was monitored. Animals were sacrificed when tumors reached 200 mm2 or for moribund states. Statistical analysis was performed in GraphPad Prism (*, p< 0.05; **, p< 0.01; ***, p< 0.001).

Results, Conclusions, and Discussions: Co-delivery of a plasmid encoding 4-1BBL with various plasmids encoding signal 3 molecules led to diverse responses in B16F10 tumors in vivo, with 4-1BBL/IL-2 and 4-1BBL/IL-12 NPs leading to the greatest reduction in tumor growth, and the highest MHC-I induction and CD8+ T-cell infiltration (Figure 1A). Across all NP formulations tested, tumor growth correlated negatively with MHC-I induction (Figure 1A, R2 = 0.501, p < 0.0001) and CD8+ T-cell infiltration (Figure 1B, R2 = 0.363, p = 0.0009). 4-1BBL/IL-2 and 4-1BBL/IL-12 NPs recruited similar percentages of CD8+ T cells to the tumor (Figure 1B); these cells showed similar levels of exhaustion, though cells recruited with 4-1BBL/IL-12 NPs were more functional than those recruited by 4-1BBL/IL-2 NPs (Figure 2). Based on these results, NPs co-delivering plasmids encoding various signal 2 molecules and IL-12 were tested in B16F10 tumors. All NP formulations drove increased CD8+ T-cell infiltration and MHC-I induction, and select formulations led to decreased tumor growth on day 20 (Figure 3A-C). To assess overall efficacy of the leading combinations, control NPs, 4-1BBL/IL-12 NPs, and CD86/IL-12 NPs were assessed in a B16F10 survival study. Tumor size was significantly lower with 4-1BBL/IL-12 and CD86/IL-12 NPs compared to control NPs on days 13 and 15, and both therapeutic NP formulations led to significant improvement in survival compared to control NPs (Figure 4A-B). Together, these results demonstrate the utility of a modular NP design for systematic screening of therapeutic plasmid delivery to melanoma in vivo, which led to the identification and profiling of top NP formulations that generate productive local anti-tumor immune responses.

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