{
  "abstract": "Background ROR1 is an attractive tumor associated antigen for cancer therapies, owing to its abundant expression in various malignancies and minimal presence in normal tissues. Here, we present a novel tri-specific antibody, TJ-RO4B, which specifically targets two distinct epitopes of ROR1 in a bi-paratopic design and the CRD3/4 domain of 4-1BB designed to achieve ROR1-dependent conditional activation of 4-1BB for enhanced T cell stimulation only within tumor microenvironment. In the form of TJ-RO4B, the 4-1BB arm does not induce the known dose-limiting toxicities, i.e. hepatotoxicity and cytokine release in association with 4-1BB’s systemic exposure. TJ-RO4B has been shown to exhibit superior and durable anti-tumor activity that is enabled by a bi-paratopic mechanism of ROR1 antibody coupled with an intact Fc region in sync with its tumor-localized 4-1BB activity. TJ-RO4B is currently in development as a promising candidate for cancer therapy.Methods The 4-1BB properties of TJ-RO4B were validated in vitro through a 4-1BB signaling reporter assay and related T cell functional assays. The Fc effector functions were assessed by ADCC and ADCP assays. In vivo efficacy and safety of TJ-RO4B were examined in 4-1BB humanized mice. Characterization of intra-tumoral and peripheral immune cells was analyzed by flow cytometry.Results TJ-RO4B was engineered as a bi-paratopic Y-body-like antibody, incorporating two non-competing anti-ROR1 domains with anti-4-1BB VHHs fused to the Fc C-terminus. Compared to a reference ROR1×4-1BB bispecific antibody that targets a single epitope, TJ-RO4B exhibited superior binding affinity to ROR1 and enhanced conditional activation of 4-1BB across a range of ROR1-expressing tumor cells, which is attributable to its bi-paratopic antibody design. Furthermore, the intact hIgG1 Fc of TJ-RO4B enabled potent ADCC and ADCP against ROR1 + or 4-1BB+ cells in vitro while no 4-1BB activation was observed with FcγR2b crosslinking. The intact Fc of TJ-RO4B exhibited increased anti-tumor activity and significant Treg depletion in tumors. In a 4-1BB humanized syngeneic mouse model, TJ-RO4B displayed potent anti-tumor effects in a dose-dependent manner, which was accompanied by increased T-cell infiltration and depletion of 4-1BBhi Tregs in tumor. In addition, TJ-RO4B demonstrated an excellent safety profile with a NOAEL at 30 mg/kg and a typical mAb-like PK profile.Conclusions The present study has provided compelling evidence that the novel tri-specific TJ-RO4B with the design of bi-paratopic ROR1 antibody and conditional activation of 4-1BB agonist antibody enables robust 4-1BB activation in a strictly ROR1-dependent and tumor-localized manner. TJ-RO4B is a promising drug candidate for immunotherapy targeting ROR1-positive malignancies.",
  "authors": [
    {
      "affiliations": [
        "TJ Biopharma, Shanghai, China"
      ],
      "name": "Chanjuan Liu"
    },
    {
      "affiliations": [
        "TJ Biopharma, Shanghai, China"
      ],
      "name": "Xiaojun Xing"
    },
    {
      "affiliations": [
        "TJ Biopharma, Shanghai, China"
      ],
      "name": "Yuan Meng"
    },
    {
      "affiliations": [
        "TJ Biopharma, Shanghai, China"
      ],
      "name": "Jingwu Zang"
    },
    {
      "affiliations": [
        "TJ Biopharma, Shanghai, China"
      ],
      "name": "Xi Chen"
    }
  ],
  "title": "965 A novel bi-paratopic ROR1×4–1BB tri-specific antibody for enhanced anti-tumor immunity through tumor-localized 4–1BB agonist activity",
  "uid": "46d5cb01-e1f1-5c7b-8a6f-9c72e15e7a47"
}