1410 Novel high-plex CODEX panel reveals induced immune changes from signal 2/3 gene delivery to murine melanoma

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Background CODEX is an emerging high-plex technique that allows the visualization of protein biomarkers while maintaining tissue architecture. Few antibodies are available to study murine formalin-fixed paraffin-embedded (FFPE) tissues, which offer several advantages over fresh-frozen tissue with respect to ease of handling/storage and the ability to design tissue microarrays (TMAs).1 To address this gap, we designed, optimized, and validated a FFPE CODEX panel. We have previously developed gene delivery nanoparticles for intratumoral co-delivery of costimulatory molecule 4-1BBL and immunostimulatory cytokine IL-12.2 3 Here, we assessed co-delivery of IL-12 with additional costimulatory molecules, CD80 and OX40L, aimed at improved immune activation against murine melanoma and applied our CODEX panel to study the induced changes in the tumor immune microenvironment (TIME). Methods C57BL/6J mice bearing B16F10 flank tumors received intratumoral injections of poly(beta-amino ester)-based NPs containing various plasmids, including luciferase (as a negative control), 4-1BBL/IL-12, CD80/IL-12, or OX40L/IL-12. Tumors were harvested and fixed, then assembled into a TMA. FFPE sections were stained with a custom-conjugated antibody panel containing 25+ antibodies (table 1), imaged on the PhenoCycler-Fusion 2.0, and analyzed in HALO. Tumor annotations were drawn around SOX10+ (melanoma) cells, followed by cell segmentation and marker thresholding. Phenotyping was conducted in R using Seurat, and spatial analysis was performed using SPIAT figure 1). Results Tumor microenvironments were markedly inflamed following administration of the three immunostimulatory NP formulations (figure 2). These formulations reduced the proportion of tumor cells (figure 3A) and increased the proportion of intratumoral immune cells (figure 3B). 4-1BBL/IL-12 NPs consistently increased the proportion of CD4 T cells (p=0.0090), CD8 T cells (p=0.0120), and NK cells (p=0.0167; figure 3C-E). Both 4-1BBL/IL-12 and OX40L/IL-12 NPs significantly increased the proportion of macrophages (figure 3F) and polarized the intratumoral macrophages towards M1 (F4/80+CD86+) from M2 (F4/80+CD163+, F4/80+CD206+, or F4/80+CD163+CD206+) (figure 3G-H). Following cell clustering and phenotyping (figure 4A), spatial analysis was performed to evaluate the local immune landscape around tumor cells. The local microenvironment within 100 um of tumor cells demonstrated a diversity of surrounding immune cells, with consistent significant increases in the proportion of CD8 T cells across all three groups (figure 4C-E). Conclusions Delivery of 4-1BBL/IL-12, CD80/IL-12, and OX40L/IL-12 reprogramming NPs to murine melanoma resulted in a proinflammatory TIME with significant T cell infiltration and M1 macrophage polarization, supporting their preclinical potential as candidates for immunotherapy development. References Black S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM, Nolan GP. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat Protoc 2021;16:3802–3835. Tzeng SY, Patel KK, Wilson DR, Meyer RA, Rhodes KR, Green JJ. In situ genetic engineering of tumors for long-lasting and systemic immunotherapy. Proc. Natl. Acad. Sci. U. S. A 2020;8:4043–4052. Luly KM, Green JJ, Sunshine JC, Tzeng SY. Biomaterial-mediated genetic reprogramming of merkel cell carcinoma and melanoma leads to targeted cancer cell killing in vitro and in vivo. ACS Biomater. Sci. Eng 2023;11:6438–6450.

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