{
  "abstract": "Background Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. Overall, ICI treatment only induces durable responses in less than 20% of cancer patients.Methods To enhance the anti-tumor activity of pembrolizumab, a PD-1 inhibitor which is used in cancer immunotherapy, we used our novel proprietary soluble human TCRβ constant domain (TRBC)-based scaffold technology to construct a novel heterodimeric tetrafunctional fusion molecule, designated as HCW11-040, which comprises IL-7, IL-15/IL-15 receptor α complex, the extracellular domains of the human transforming growth factor-β receptor RII (TGFβRII) (TGFβ trap) and the antigen-binding domain of pembrolizumab in a diabody format (DbαPD1). HCW11-040 was expressed in CHO cells and purified using anti-TRBC antibody-based affinity chromatography. The presence of IL-7, IL-15 and TGFβRII domains of HCW11-40 were confirmed with ELISA based on binding activities to correspondent antibodies while PD1 binding of DbαPD1 was demonstrated using biotinylated PD1-Fc fusion protein. The biological activities of IL-7, IL-15 and TGFβ trap of HCW11-040 were evaluated using their specific report cell lines. The blocking activity of PD1-PDL1 interaction by HCW11-040 was evaluated with an anti-immune checkpoint cell-based assay (InvivoGen). For in vitro activation assay, human PBMCs were cultured with HCW11-040 (100nM) for 3-6 days and the cell phenotypes were analyzed. For in vitro cytotoxicity assay, the activated human PBMC were co-cultured with SW1990 pancreatic cell lines and analyzed by flow cytometry utilizing Propidium Iodide dye to identify dead SW1990 cells.Results HCW11-040 exhibits molecular weight 347.5 kDa by size exclusion chromatography. The biological activity assay of functional domains of HCW11-040 showed that EC50 was 25 pM for IL-7, 108 pM for IL-15 and 424 pM for TGFβ trap. The blocking activity of PD1-PDL1 interaction by HCW11-040 was comparable with commercially available full-length pembrolizumab (SelleckChem) based on an anti-immune checkpoint cell-based assay. In vitro, HCW11-040 more potently activates human CD4+ T cells, CD8+ T cells and natural killer (NK) cells, promotes their proliferation and enhances their cytotoxicity against human SW1990 pancreatic tumor cell line when compared to pembrolizumab. HCW11-040 was also found to neutralize the immunosuppresiveness of TGF-β1. Extensive studies are being conducted to evaluate its anti-tumor activities in comparison with pembrolizumab in animal models.Conclusions HCW11-040 may serve as a novel therapeutic to simultaneously work as an immune checkpoint inhibitor, provide immunostimulation of immune cells and lessen immunosuppression associated with tumor microenvironment of cancer.",
  "authors": [
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Bai Liu"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Lin Kong"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Lucas Gomez"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Lijing You"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Jilan Xing"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Neicy Gonzalez"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Zheng Wang"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Xiaoyun Zhu"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Varghese George"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Christian Echeverri"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Niraj Shrestha"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Liying Li"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Jack O Egan"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Peter R Rhode"
    },
    {
      "affiliations": [
        "HCW Biologics Inc. Miramar, FL, USA"
      ],
      "name": "Hing C Wong"
    }
  ],
  "title": "1014 A novel tetra-specific pembrolizumab-based immunotherapeutic",
  "uid": "6c997452-dcc1-5ff2-8cf1-08f645e4cc27"
}