26 High-plex immunofluorescence imaging yields insights into antigen presentation and checkpoint expression following nanoparticle reprogramming of murine melanoma

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Background We have previously shown that 4-1BBL/IL-12 nanoparticles (NPs) are capable of eliciting potent immune responses in B16F10 melanoma tumors.1 We further showed that incorporation of IFNγ plasmid drove higher levels of MHC-I expression in vitro compared to 4-1BBL/IL-12 or control Nps.2 Despite this, when combined with anti-PD1 in vivo, addition of IFNγ did not result in improved survival and led to an exhausted CD8 T-cell phenotype.2 3 Here, we apply our novel high-plex immunofluorescence panel to characterize the induced tumor microenvironment (TME) following NP immunoengineering, yielding insights into why the addition of IFNγ did not result in improved tumor control. Methods Poly(beta-amino ester)-based NPs were formulated with plasmids encoding luciferase, 4-1BBL/IL-12, or 4-1BBL/IL-12/IFNγ. C57BL/6J mice bearing B16F10 flank tumors received intratumoral NP injections and intraperitoneal injections of anti-PD1 on days 9, 11, 16, and 18. On day 20, tumors were harvested and fixed. Formalin-fixed paraffin-embedded samples were sectioned and stained with a DNA barcode custom-conjugated antibody panel and imaged on the PhenoCycler-Fusion 2.0. QPTIFF files were imported into the digital pathology suite HALO for cell segmentation, marker thresholding, and mean fluorescence intensity (MFI) calculation (figure 1). Tumor cells (SOX10+F4/80-CD11c-), macrophages (F4/80+), and dendritic cells (CD11c+) were identified. Antigen presentation was characterized using LMP2 and beta 2-microglobulin (β2M), and PD-L1 expression was evaluated. Results 4-1BBL/IL-12 NPs with anti-PD1 increased the proportion of LMP2-expressing tumor cells by 2.6-fold and LMP2 MFI by 1.4-fold over luciferase NPs (figures 2, 3A, 3D). 4-1BBL/IL-12/IFNγ NPs with anti-PD1 consistently increased the proportion of β2M-expressing tumor cells, macrophages, and dendritic cells, and significantly increased β2M MFI by 2.4-fold (p=0.017; figures 3E-H). Additionally, the 4-1BBL/IL-12/IFNγ regimen significantly increased the proportion of PD-L1-expressing tumor cells (p=0.008) and macrophages (p=0.016), whereas the 4-1BBL/IL-12 regimen significantly increased the proportion of PD-L1-expressing macrophages (p=0.028) and dendritic cells (p=0.040; figures 4, 5A-C). Both combinations significantly increased PD-L1 MFI relative to luciferase NPs: 4-1BBL/IL-12 NPs with anti-PD1 caused a 2.0-fold increase (p=0.046) and 4-1BBL/IL-12/IFNγ NPs with anti-PD1 caused a higher (2.5-fold) increase (p=0.005; figure 5D). Conclusions When administered with systemic anti-PD1, 4-1BBL/IL-12 and 4-1BBL/IL-12/IFNγ NPs differentially upregulate expression of antigen presentation markers in the melanoma TME, with 4-1BBL/IL-12 inducing higher expression on tumor cells and the addition of IFNγ resulting in higher expression on professional antigen presenting cells. Furthermore, 4-1BBL/IL-12/IFNγ NPs cause a greater increase in the expression of immunologic checkpoint PD-L1, offering a potential additional explanation for the reduced efficacy of 4-1BBL/IL-12/IFNγ NPs in vivo. References 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;117(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;9(11):6438–6450. Luly KM, Green JJ, Tzeng SY, Sunshine JC. 222 Genetic reprogramming of merkel cell carcinoma and melanoma leads to increased MHC-I expression and antitumor immune activation in vitro and in vivo. J Immunother Cancer 2021;9.

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