{
  "abstract": "Background Bispecific T cell engagers have demonstrated promising clinical progress in both hematologic malignancies and solid tumors; however, their therapeutic efficacy is still limited by multiple challenges including T cell anergy resulting from single-signal activation exclusively through CD3 engagement. As a critical co-stimulatory molecule, CD28 enhances T-cell functionality through synergistic interaction with the TCR-mediated primary activation signal, thus potentiating antitumor efficacy. Non-superagonistic CD28 bispecific antibodies, while enhancing antitumor efficacy, can minimize systemic toxicity. Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1), a cancer-testis antigen overexpressed in diverse malignancies, emerges as a promising target for tumor-specific immunotherapy. Herein, the development of a dual-signal T-cell engager strategy targeting KK-LC-1, using a non-superagonist CD28-based co-stimulatory mechanism, is of critical importance.Methods Based on the successful acquisition of the designed ankyrin repeat proteins targeting KK-LC-1 and CD28 through phage display technology (KD=8.985 nM and 7.43 nM), we designed two T cell engagers (KK-LC-1×CD3 and KK-LC-1×CD28). The tumor-specific binding activity of KK-LC-1 designed ankyrin repeat protein was verified by surface plasmon resonance, flow cytometry, confocal microscopy, and in vivo imaging. We then fused it with CD3 single-chain variable fragment and CD28-designed ankyrin repeat protein, respectively, to construct two T cell engagers. Their biological activities and antitumor efficacy were systematically evaluated both in vitro and in vivo (n=5) using flow cytometric analysis, confocal microscopy imaging, and bioluminescence quantification.Results T-cell engagers KK-LC-1×CD3 and KK-LC-1×CD28 were successfully engineered and demonstrated high binding affinity for both KK-LC-1-positive tumor cells and T cells. Co-administration of these engagers significantly augmented T-cell activation and antitumor efficacy (88% vs 66%, p<0.001) compared with KK-LC-1×CD3 monotherapy. In vivo, the combination suppressed tumor growth by 59.6% vs monotherapy (p<0.05) with enhanced intratumoral CD8 + infiltration (5.4-fold, p<0.001) and CD4+ infiltration (2.7-fold, p<0.001), while triple therapy incorporating PD-1×CTLA-4 bispecific antibodies extended median survival from 44 to 48 days (p<0.05).Conclusions We validated the feasibility of the KK-LC-1-targeted dual-signal T-cell engager strategy for the treatment of solid tumors and demonstrated that its combination with PD-1×CTLA-4 bispecific antibodies synergistically enhanced antitumor efficacy in preclinical studies.",
  "authors": [
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Tianran Chen"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Ying Wang"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Xiaotong Chen"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Yuxiang Li"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Jingyi Guo"
    },
    {
      "affiliations": [
        "Department of Pathology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China"
      ],
      "name": "Fangcen Liu"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Jie Shao"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Jiayao Yan"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Mengzhu Li"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Jiaqi Nie"
    },
    {
      "affiliations": [
        "Department of Pathology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China"
      ],
      "name": "Qi Sun"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Qin Liu"
    },
    {
      "affiliations": [
        "The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital & Group’s Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China",
        "Clinical Cancer Institute of Nanjing University, Nanjing, China"
      ],
      "name": "Baorui Liu"
    }
  ],
  "title": "Non-superagonist CD28-based dual-signal T cell engager targeting KK-LC-1 enhances antitumor efficacy",
  "uid": "f7717ce1-1c76-5335-82ef-042312cf320c"
}