{
  "abstract": "Background Cold solid tumors, including pancreatic, prostate, and microsatellite stable colorectal cancers, remain resistant to immunotherapy due to immune excluded microenvironments. 1 2 These tumors exhibit minimal cytotoxic T-cell infiltration, impaired antigen presentation, and a suppressive cytokine profile that hinder immune recognition and limit the efficacy of therapies reliant on T-cell activity, such as PD-1 checkpoint blockade.1 Immune stimulatory agents interleukin-2 (IL-2) and the microbial metabolite butyrate are under investigation for tumor microenvironment modulation. IL-2 promotes expansion and activation of cytotoxic T cells and natural killer (NK) cells, while butyrate enhances antigen presentation.3 4 However, IL-2’s systemic toxicity, poor tumor specificity, and butyrate’s instability and non-targeted distribution have hindered clinical translation.3 5 These limitations highlight the need for an integrated strategy capable of localizing immune activation and metabolic support within cold tumors. A tri-specific fusion antibody is proposed that combines PD-1 blockade, localized IL-2 stimulation, and selective butyrate retention to restore immune visibility and responsiveness in immune excluded tumors.Methods A tri-specific single chain fusion antibody is proposed, consisting of three functional domains connected by flexible peptide linkers: a PD-1 binding single chain variable fragment (scFv) domain designed to inhibit PD-1 mediated immune suppression, an engineered interleukin-2 (IL-2) domain designed to preferentially activate CD8 + T cells and natural killer (NK) cells while minimizing systemic toxicity through immune cell anchored delivery, and a butyrate binding domain designed to retain microbiota derived butyrate within the tumor microenvironment. The construct is envisioned for tumor localized delivery to enable coordinated checkpoint inhibition, immune cell stimulation, and metabolic support within immune excluded tumors such as pancreatic, prostate, and microsatellite stable colorectal cancers.Results The proposed tri-specific antibody is expected to enhance antitumor immunity by promoting intratumoral CD8 + T-cell and natural killer (NK) cell infiltration, increasing effector cytokine production (e.g., IFN-γ, TNF-α), and reducing immunosuppressive regulatory T-cell (Treg) populations. By integrating checkpoint inhibition, targeted IL-2 stimulation, and metabolic reprogramming via butyrate retention, this strategy may overcome immune exclusion in cold tumors and demonstrate superior efficacy compared to PD-1 monotherapy or current bispecific formats. These anticipated outcomes remain to be validated in preclinical tumor models.Conclusions This tri-specific fusion antibody represents a rational strategy to overcome immune exclusion in cold tumors. By restoring immune visibility and effector function within the tumor microenvironment, it offers a mechanistically grounded path toward enhanced responsiveness in resistant cancers. Preclinical and computational validation will be essential for advancing clinical translation.References Ju Y, Xu D, Liao MM, Sun Y, Bao WD, Yao F, Ma L. Barriers and opportunities in pancreatic cancer immunotherapy . NPJ Precis Oncol. 2024;8:199.Zhang D, Ni QQ, Liang QY, He LL, Qiu BW, Zhang LJ, et al. ASCL2 induces an immune-excluded microenvironment by activating cancer-associated fibroblasts in microsatellite-stable colorectal cancer. Oncogene. 2023;42:2841–2853.Raker VK, Becker C, Landfester K, Steinbrink K. Targeted activation of T cells with IL-2-coupled nanoparticles. Cells. 2020;9:2063.Perrin P, Cassagnau E, Burg C, Patry Y, Vavasseur F, Harb J, et al. An interleukin 2/sodium butyrate combination as immunotherapy for rat colon cancer peritoneal carcinomatosis. Gastroenterology. 1994;107:1697–1708.Siddiqui MT, Cresci GAM. The immunomodulatory functions of butyrate. J Inflamm Res. 2021;14:6025–6041.",
  "authors": [
    {
      "affiliations": [
        "University of Hertfordshire, Sharqia, Egypt"
      ],
      "name": "Moaz E Sayed Ahmed"
    }
  ],
  "title": "1022 Engineering a tri-specific antibody targeting PD-1, IL-2, and butyrate to reprogram cold tumors",
  "uid": "71f6d915-075e-53ee-af29-23f79d9000c5"
}