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<p>Feline lymphoma, a prevalent feline malignancy, responds poorly to conventional chemotherapy—a setting that underscores the urgent need for targeted therapeutic alternatives. The stable expression of CD3 on T-cell lymphoma cells positions this molecule as an ideal immunotherapeutic target. Compared with conventional antibodies, single-domain nanobodies derived from camelid heavy-chain antibodies offer distinct advantages, including smaller size, enhanced tissue penetration, and superior stability, thereby rendering CD3-targeted nanobodies particularly promising for managing feline T-cell lymphoma. We immunized a Bactrian camel with recombinant feline CD3ε extracellular domain and constructed a phage display nanobody library (capacity 5.97 × 10<sup>8</sup>). Sequencing of the constructed library revealed high diversity; among 10 randomly selected clones, 7 unique sequences were identified, and 93.33% of the clones carried intact VHH inserts. After three panning rounds, three high-affinity single-domain antibodies were selected and expressed, with clone 73 retaining stable binding at 156&#xa0;ng/mL concentration. Flow cytometry showed that clone 73 specifically recognized approximately 37.95% of feline peripheral blood CD3<sup>+</sup> cells. Western blotting detected a specific ~ 20&#xa0;kDa band in feline lymphocyte lysates, and immunofluorescence confirmed precise cellsurface localization without non-specific signal. These findings demonstrate successful generation of feline CD3-specific nanobodies, which enable flow cytometric diagnosis of feline T-cell lymphoma and provide a foundation for developing immunotherapeutics.</p>

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Preparation, characterization and application of feline CD3-specific single-domain antibodies

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月7日

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  • Jingjing Wang,
  • Qihuan Zhao,
  • Bo Wang,
  • Chengquan Du,
  • Hui Wang,
  • Jingsi Mei,
  • Ruifeng Gao,
  • Fuxiang Bao

摘要

Feline lymphoma, a prevalent feline malignancy, responds poorly to conventional chemotherapy—a setting that underscores the urgent need for targeted therapeutic alternatives. The stable expression of CD3 on T-cell lymphoma cells positions this molecule as an ideal immunotherapeutic target. Compared with conventional antibodies, single-domain nanobodies derived from camelid heavy-chain antibodies offer distinct advantages, including smaller size, enhanced tissue penetration, and superior stability, thereby rendering CD3-targeted nanobodies particularly promising for managing feline T-cell lymphoma. We immunized a Bactrian camel with recombinant feline CD3ε extracellular domain and constructed a phage display nanobody library (capacity 5.97 × 108). Sequencing of the constructed library revealed high diversity; among 10 randomly selected clones, 7 unique sequences were identified, and 93.33% of the clones carried intact VHH inserts. After three panning rounds, three high-affinity single-domain antibodies were selected and expressed, with clone 73 retaining stable binding at 156 ng/mL concentration. Flow cytometry showed that clone 73 specifically recognized approximately 37.95% of feline peripheral blood CD3+ cells. Western blotting detected a specific ~ 20 kDa band in feline lymphocyte lysates, and immunofluorescence confirmed precise cellsurface localization without non-specific signal. These findings demonstrate successful generation of feline CD3-specific nanobodies, which enable flow cytometric diagnosis of feline T-cell lymphoma and provide a foundation for developing immunotherapeutics.