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<p>Colorectal cancer (CRC) is the second leading cause of cancer-related mortality, and the incidence of early-onset colon cancer has been increasing globally in recent years. The development of immunotherapies for colon cancer is critical for providing new treatment strategies to combat drug resistance. Here, a bispecific nanobody against PD-L1 (BsNb-PD-L1) constructed using genetically encoded noncanonical amino acids (ncAAs) is reported. A computational protocol was developed to identify appropriate sites in the nanobody for incorporating p-acetylphenylalanine (pAcF). Variants of nanobodies PV2 and PV3 with pAcF incorporated were conjugated with linkers containing an aminooxy functionality to enable oxime ligation. The resulting PV2-S71 + PV3-N77 bispecific nanobody (BsNb-ncAA) exhibited higher thermostability and binding affinity compared to the nanobody monomers and the BsNb constructed by simply fusing two proteins. Moreover, in an in vitro phagocytosis model, the BsNb-ncAA exhibited improved capability to inhibit immune evasion and showed stronger biological activity compared to the fusion protein PV2-PV3. Furthermore, the BsNb-ncAA resulted in a marked increase in the number of CD8<sup>+</sup> T cells within tumor tissues and demonstrated efficient inhibitory effects against colon tumor growth in vivo. Our study provides a general strategy for constructing BsNbs, which has potential applications in other cancer immunotherapy.</p><p></p>

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Noncanonical amino acid-aided synthesis of anti-PD-L1 bispecific nanobody for colon cancer immunotherapy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月24日

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  • Xinyu Hu,
  • Ling Jiang,
  • Haibin Yuan,
  • Wanyi Chen,
  • Yufei Guo,
  • Mengxue Gao,
  • Yijia Song,
  • Zichen Wang,
  • Yuli Wang,
  • Miao Zhang,
  • Ario de Marco,
  • He Huang,
  • Guangbo Kang,
  • Haoran Yu

摘要

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality, and the incidence of early-onset colon cancer has been increasing globally in recent years. The development of immunotherapies for colon cancer is critical for providing new treatment strategies to combat drug resistance. Here, a bispecific nanobody against PD-L1 (BsNb-PD-L1) constructed using genetically encoded noncanonical amino acids (ncAAs) is reported. A computational protocol was developed to identify appropriate sites in the nanobody for incorporating p-acetylphenylalanine (pAcF). Variants of nanobodies PV2 and PV3 with pAcF incorporated were conjugated with linkers containing an aminooxy functionality to enable oxime ligation. The resulting PV2-S71 + PV3-N77 bispecific nanobody (BsNb-ncAA) exhibited higher thermostability and binding affinity compared to the nanobody monomers and the BsNb constructed by simply fusing two proteins. Moreover, in an in vitro phagocytosis model, the BsNb-ncAA exhibited improved capability to inhibit immune evasion and showed stronger biological activity compared to the fusion protein PV2-PV3. Furthermore, the BsNb-ncAA resulted in a marked increase in the number of CD8+ T cells within tumor tissues and demonstrated efficient inhibitory effects against colon tumor growth in vivo. Our study provides a general strategy for constructing BsNbs, which has potential applications in other cancer immunotherapy.