De novo design of a two-step approach targeting Claudin-6 for enhanced drug delivery to solid tumors

  • J Transl Med. 2025 Nov 20;23(1):1323. doi: 10.1186/s12967-025-07316-2.
Jiayao Yan  #  1 ,  Liqing Zhong  #  1 ,  Xiaotong Chen  1 ,  Lin Li  2 ,  Fangcen Liu  2 ,  Lei Lei  3 ,  Mengchao An  4 ,  Xiao Wei  2 ,  Ying Wang  1 ,  Tianran Chen  1 ,  Jingyi Guo  5 ,  Jie Shao  1 ,  Xiaoxiao Yu  6 ,  Yingjie Zhao  7 ,  Rutian Li  8 ,  Qin Liu  9  10 ,  Baorui Liu  11  12  13  14
Affiliations
  • 1. Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China.
  • 2. Department of Pathology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China.
  • 3. Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.
  • 4. The Comprehensive Cancer Center, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, China.
  • 5. Department of Oncology, Nanjing Drum Tower Hospital, China Pharmaceutical University, Nanjing, China.
  • 6. School of Life Sciences, Nanjing University, Nanjing, China.
  • 7. Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • 8. Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China. [email protected].
  • 9. Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China. [email protected].
  • 10. Department of Oncology, Nanjing Drum Tower Hospital & Group's Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China. [email protected].
  • 11. Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China. [email protected].
  • 12. The Comprehensive Cancer Center, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, China. [email protected].
  • 13. Department of Oncology, Nanjing Drum Tower Hospital, China Pharmaceutical University, Nanjing, China. [email protected].
  • 14. Department of Oncology, Nanjing Drum Tower Hospital & Group's Suqian Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China. [email protected].
  • # Contributed equally.
PMID: 41267074 DOI: 10.1186/s12967-025-07316-2
Abstract

Background: Although antibody-conjugated drugs have achieved success in clinical practice for Cancer treatment, challenges remain in developing a highly efficient drug delivery system with specific accumulation in tumors and reduction in side effects. With improved pharmacokinetics, strong covalent bonding and quick binding reactions, a pre-targeting approach via molecular pairs represents an attractive platform for two-step delivery system construction.

Methods: Bioinformatics and immunohistochemistry assays were performed to assess Claudin-6 (CLDN6) as a highly specific tumor target in solid tumors. A phage-displayed library was used to screen and optimize anti-CLDN6 designed ankyrin repeat proteins (DARPins), which were incorporated into a two-step delivery system based on SpyTag/SpyCatcher. Fluorescent staining, flow cytometry and near-infrared imaging were performed to assess the tumor-targeting ability and biodistribution of this delivery system. The cytotoxic drug, Monomethyl Auristatin E (MMAE), was conjugated with the delivery system to evaluate its anti-tumor efficacy and safety profile.

Results: Anti-CLDN6 DARPins exhibited specific binding to CLDN6+ Cancer cells with high affinity instead of negative cells in vitro, ex vivo and in vivo. The DARPins-based two-step delivery system improved background clearance with a high signal-to-noise ratio, enhancing the specific accumulation of payloads in tumors. The cytotoxic drug delivered via the two-step system appeared superior to the one-step approach in IC50, biodistribution, and tumor growth inhibition.

Conclusions: Our study presented the de novo design of a two-step drug delivery system targeting Claudin-6 with enhanced anti-tumor efficacy and improved biosafety. These findings highlighted the potential of this approach to enhance the efficacy of tumor-targeting therapies and reduce adverse effects, paving the way for more effective Cancer treatments.

Keywords
CLDN6; DARPins; Phage display; Pre-targeting.
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