{
  "abstract": "Background PD-1, CTLA-4, and VEGF are validated clinical targets with complementary roles in suppressing anti-tumor immunity. PD-(L)1/VEGF and PD-(L)1/CTLA-4 bispecific antibodies show promising clinical benefits for certain solid tumors, and several on-going studies explore a tri-targeting multi-drug combination approach. Given the avidity and co-operative binding advantages conferred on multispecific antibodies, combining anti-PD-1 (restore effector T cell activity), anti-CTLA4 (T cell priming/proliferation), and anti-VEGF (inhibit tumor angiogenesis) may provide better biological synergy and clinical benefits. In this study, CT111, our lead PD-1 x CTLA-4 x VEGF trispecific antibody, was characterized to gain mechanistic insights into its functional attributes in synergistically blocking PD-1/CTLA-4-mediated immunosuppression and VEGF-induced tumor angiogenesis.Methods CT111 consists of VHHs targeting PD-1, CTLA-4, and VEGF as a hexavalent Fc-silenced homodimer. In vitro activity on individual targets and avidity-based synergistic effect were thoroughly evaluated in multiple assays. Preclinical efficacy was evaluated in transgenic hPD-1/hPD-L1/hCTLA-4-BALB/c mice implanted with CT26-hVEGF/hPD-L1 tumors and HSC humanized HCC827 tumor model.Results CT111 exhibited strong binding affinity to human and cynomolgus targets and potently blocked ligand-receptor interactions, outperforming clinical benchmarks. Potent anti-PD-1/anti-CTLA-4 synergy was observed with CT111 compared to AK104 in SEB-stimulated primary immune cells, as evidenced by high IL-2 secretion and T-cell proliferation across multiple human donors. Cooperative binding to VEGF significantly enhanced binding affinities on PD-1 and CTLA-4 expressing CHO cells and activated human T cells. Increased avidity was concordantly reflected functionally with significant enhancement of CD80 and PD-L2 ligand blocking activity on CTLA-4 and PD-1 expressing cells. In preclinical models, CT111 treatment at low doses elicited potent anti-tumor activity against CT26-hVEGF/hPD-L1 tumors in BALB/c hPD-1/hPD-L1/hCTLA-4 mice. Potent and sustained tumor regression was similarly observed with low doses of CT111 with superior efficacy compared to clinical benchmark antibodies in the HSC humanized HCC827 NSCLC tumor model. Analysis of HCC827 tumors revealed significant TME changes with reduced tumor vascularization, increased number of activated CD8+ T cells and decreased Tregs. Notably, the lack of Fc-effector functions in CT111 suggests intratumoral Treg reduction is not attributed to Fc-mediated Treg depletion, but rather downstream effects of blocking CTLA-4 signaling.Conclusions CT111 mediates strong synergy to re-invigorate T cell proliferation and anti-tumor response, while effectively blocking VEGF-induced tumor angiogenesis. Cooperative binding to VEGF in vitro significantly enhances avidity for PD-1 and CTLA-4, suggesting that tumor-secreted VEGF may amplify CT111 activity within the TME. These findings support clinical development of CT111 for treatment of solid tumors, particularly in anti-PD-1 resistance setting.",
  "authors": [
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "John Lee"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Leo Ng"
    },
    {
      "affiliations": [
        "Shanghai Cell Therapy Group Co. Ltd, Shanghai, China"
      ],
      "name": "Jing Yu"
    },
    {
      "affiliations": [
        "Shanghai Cell Therapy Group Co. Ltd, Shanghai, China"
      ],
      "name": "Jia Yu"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Huiyuan Tang"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Weihong Wang"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Yong Wang"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Toya N Baral"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Xinhao Wang"
    },
    {
      "affiliations": [
        "Shanghai Cell Therapy Group Co. Ltd, Shanghai, China"
      ],
      "name": "Jiaguo Li"
    },
    {
      "affiliations": [
        "Shanghai Cell Therapy Group Co. Ltd, Shanghai, China"
      ],
      "name": "Weimin Zhu"
    },
    {
      "affiliations": [
        "Shanghai Cell Therapy Group Co. Ltd, Shanghai, China"
      ],
      "name": "Qijun Qian"
    },
    {
      "affiliations": [
        "Chantibody Therapeutics, Inc., Menlo Park, CA, USA"
      ],
      "name": "Wenfeng Xu"
    }
  ],
  "title": "976 CT111, a PD-1 x CTLA-4 x VEGF trispecific antibody, demonstrates potent anti-tumor efficacy through synergistic immune checkpoint blockade and VEGF inhibition with cooperative avidity",
  "uid": "60cd9968-5f98-5cb7-92b0-43ed552247d3"
}