{
  "abstract": "Background Immune checkpoint inhibitors (ICIs), such as αPD(L)1 antibodies, have been widely approved for several cancers, but their efficacy is limited to specific cancer types, primarily due to insufficient activation of immune cells in tumor microenvironment (TME). Combination with αPD-1 and αVEGF monoclonal antibodies (mAbs) or bispecific antibodies (bsAbs) have demonstrated improved efficacy compared to αPD-1 monotherapy across various cancer types. Despite such advances, boosting immune cell proliferation within the TME remains crucial for achieving superior efficacy in ICI-resistant or refractory patients. Interleukin-2 (IL-2) is a potent T cell modulator capable of overcoming these limitations, but the severe systemic toxicity of IL-2 limits its clinical application, prompting the development of IL-2 variants (IL-2v) with TME selectivity. Here, we present a highly TME selective αPD-1/αVEGF/IL-2v tri-specific fusion protein, which was engineered to continuously activate T cells via αPD-1, enhance tumor-infiltrating lymphocytes (TILs) via αVEGF, and strongly proliferate effector T cells via novel IL-2v.Methods The intrinsic activities of αPD-1/αVEGF/IL-2v to target proteins were determined by inhibitor screening assay and ELISA. TME-selective T cell activation and expansion were evaluated in hPBMC using flow cytometry. Off-target cytotoxicity was assessed in 293T cells co-cultured with hPBMC. Trans-endothelial T cell migration was assessed using HUVEC monolayer co-culture system. The anti-tumor efficacy of αPD-1/αVEGF/IL-2v was assessed in humanized mice bearing TKI-resistant lung cancer cells.Results αPD-1/αVEGF/IL-2v exhibited the intrinsic activity of its three functional arms, featuring a novel IL-2v and dual blockade of PD-1 and VEGF. Under PD-1 abundant TME-like conditions, αPD-1/αVEGF/IL-2v significantly enhanced CD8 + T cell activation, proliferation, and cytokine production compared to αPD-1 or αPD-1/αVEGF. In contrast, under peripheral blood-like conditions, CD8+ T cell responses were minimal, comparable to αPD-1 or αPD-1/αVEGF, indicating the selective activity of αPD-1/αVEGF/IL-2v in TME-mimicking environments. Furthermore, the αVEGF arm enabled the formation of drug-VEGF complexes in VEGF-rich condition, which further enhanced target engagement and functional potency. In addition, αPD-1/αVEGF/IL-2v facilitated trans-endothelial migration of T cells, supporting its potential to enrich TILs. Consistent with the in vitro results, αPD-1/αVEGF/IL-2v demonstrated superior anti-tumor efficacy without body weight loss in TKI-resistant lung cancer bearing humanized mice, compared to αPD-1 or αPD-1/αVEGF bsAbs.Conclusions αPD-1/αVEGF/IL-2v is a highly TME-selective tri-specific fusion protein designed to enhance anti-tumor efficacy with favorable tolerability. These preclinical findings suggest that αPD-1/αVEGF/IL-2v holds therapeutic potential as a next-generation immunotherapy capable of overcoming the limitations of current αPD-1 or αPD-1/αVEGF-based therapies.",
  "authors": [
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Suna Kim"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Bom Park"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Sunjung Cho"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Yongjun Jung"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Mi-Seong Kim"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Suyoon Kim"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Jun-Eui Park"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Young Jin Park"
    },
    {
      "affiliations": [
        "Mustbio, Suwon-si, Gyeonggi-do, Republic of Korea"
      ],
      "name": "Sungyoub Jung"
    }
  ],
  "title": "990 A highly TME-selective αPD-1/αVEGF/IL-2v tri-specific fusion protein demonstrates potent anti-tumor activity beyond αPD-1/αVEGF therapy",
  "uid": "f8154b90-a51b-5ec1-9396-29dd3786c6b7"
}