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

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.