{
  "abstract": "Background Loss of antigen processing machinery (APM) and downregulation of MHC-I presentation, documented in 40-90% of human tumors, is a critical tumor escape mechanism which shields cancer from recognition by the adaptive immune system. 1–4 While some studies have shown that restoring APM or master transcriptional regulators ex vivo can sensitize tumors to immune attack,5–7 obtaining optimal factor combinations and delivery of necessary genetic components remain as two critical barriers to leveraging this strategy as an in situ antigen-agnostic tumor therapy. Poly(β-amino ester) (PBAE) nanoparticles are a promising non-viral delivery system due to their tunability, biodegradability, and ability to transfect a wide range of cell types.8–10 To address these barriers, we developed a library of PBAEs and APM pDNA factors and evaluated restored antigen presentation.Methods We screened a PBAE nanoparticle library across five tumor models (MC38, CT26, LL2, 4T1, B16-F10) with varying baseline MHC expression to identify broadly applicable gene delivery formulations ( figure 1A). Lead candidates successfully transfected a panel of 11 antigen processing and transcriptional regulator genes, including NLRC5, TAP1, TAP2, Psmb9, Psmb8, IRF1, IRF2, Tapasin, Batf3, CIITA, and ERp57, into low-MHC B16-F10 and 4T1 cells, confirmed by qPCR (1C). The effect of APM delivery on surface MHC-I, MHC-II, and PD-L1 expression was evaluated via flow cytometry (figure 1B,D). To assess functional immunity, B16-F10 cells, transfected with top APM components, were co-cultured with PMEL-17 CD8+ T cells, and evaluated for antigen specific T-cell proliferation and tumor cell killing (figure 2A-B).Results Our screen identified several lead formulations, with branched polymer architectures consistently yielding the highest transfection efficiency ( figure 1A). An optimized candidate, B9,B8(30%)-S91-E1 delivered an 11-gene panel of APM and transcriptional regulators into B16-F10 and resulted in >10-fold MHC-I and >100-fold MHC-II upregulation (figure 1B). Unlike IFN and IRF1, NLRC5 notably enhanced MHC-I without increasing PD-L1 expression (figure 1D). When co-cultured with PMEL-17 antigen specific CD8+ T cells that recognize GP100 on melanoma, these engineered B16-F10 cells increased antigen specific T-cell proliferation (figure 2B) and enhanced tumor killing (figure 2A), demonstrating that APM gene delivery enhances functional tumor antigen expression and suggesting potential therapeutic benefit.Conclusions PBAE nanoparticles are a versatile platform for delivering complex genetic payloads to diverse tumors. By restoring antigen presentation machinery, we rendered immune-evasive cancer cells susceptible to T-cell mediated killing in vitro. In vivo experiments in mouse models are currently underway to evaluate the mechanism and therapeutic potential of this approach, alone or in combination with other immunotherapies.References Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, Schreiber RD. IFNγ and lymphocytes prevent primary tumour development and shape tumour immunogenicity. 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Poly(beta-amino ester)s as gene delivery vehicles: challenges and opportunities. Expert Opin. Drug Deliv. 2020;17(10):1395–1410. https://doi.org/10.1080/17425247.2020.1796628.10 Rocher EE, Luly KM, Tzeng SY, Sunshine JC, Green JJ. Efficient polymeric nanoparticle gene delivery enabled via tri- and tetrafunctional branching. Biomacromolecules. 2024;25(11):7260–7273. https://doi.org/10.1021/acs.biomac.4c00954.Abstract 1025 Figure 1100 polymers were screened to find the best for delivering immune-regulating genes to cancer cells. Top-performing PBAE NPs were used, and successful gene and protein expression (MHC-I/II, PD-L1) were confirmed via qPCR and flow cytometryAbstract 1025 Figure 2B16-F10 melanoma cells were transfected with nanoparticles carrying various APM genes. PMEL CD8+ T-cells were then co-cultured with the cancer cells at different effector-to-tumor ratios. Surviving CD8+ T-cell counts were quantified via flow cytometry",
  "authors": [
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Grace M Shoemaker"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA"
      ],
      "name": "Jack Kollings"
    },
    {
      "affiliations": [
        "Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA"
      ],
      "name": "Kevin K Zhang"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Ryan Idnani"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA",
        "Johns Hopkins University Translational Tissue Engineering Center, Baltimore, MD, USA"
      ],
      "name": "Sydney R Shannon"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Romina Blanco-Sarmiento"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Ethan Idnani"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA",
        "Johns Hopkins University Translational Tissue Engineering Center, Baltimore, MD, USA"
      ],
      "name": "Kathryn M Luly"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Lance Xu"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA",
        "Johns Hopkins University Translational Tissue Engineering Center, Baltimore, MD, USA"
      ],
      "name": "Stephany Y Tzeng"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA",
        "Johns Hopkins University Translational Tissue Engineering Center, Baltimore, MD, USA"
      ],
      "name": "Jordan J Green"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA",
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA",
        "Johns Hopkins Center for Translational ImmunoEngineering (JH-TIE), Baltimore, MD, USA",
        "Johns Hopkins University Translational Tissue Engineering Center, Baltimore, MD, USA"
      ],
      "name": "Joel C Sunshine"
    }
  ],
  "title": "1025 Engineering structurally diverse PBAE nanoparticles to restore tumor immunogenicity via antigen processing machinery pDNA delivery",
  "uid": "612c5fbf-2496-50f2-b0f7-f88cb4de6403"
}