Preparation of a camel-canine chimeric antibody against canine B-cell lymphoma

Canine B-cell lymphoma is a malignant hematologic neoplastic disease, with diffuse large B-cell lymphoma being the predominant subtype. This subtype highly expresses canine CD20 on its surface. Most clinical treatments are chemotherapeutic, with the standard treatment being the CHOP regimen. In immunotherapy, rituximab is effective for treating human B-cell lymphoma. However, its inability to bind natural canine CD20 renders it ineffective in treating canine B-cell lymphoma. The main objective of this study was to establish camel-canine chimeric antibodies and validate their in vitro biological activity. A Bactrian camel was immunized with the CD20 extracellular region peptide. The VHH fragment was amplified using nested PCR, ligated to the phage vector pMECS, and used to construct a phage antibody library with a capacity of 3.4 × 10¹⁰. After three rounds of enrichment and screening, 92 clones were selected for phage ELISA. Clones 4, 5, 8, 30, 43 and 46 exhibited high binding affinities for canine CD20. These strains were induced and purified using a prokaryotic expression system to obtain anti-canine CD20 single-domain antibodies. ELISA, Western blotting and flow cytometry analysis showed that the anti-canine CD20 single-domain antibody specifically binds to canine CD20. Cellular immunofluorescence validation revealed that the anti-canine CD20 single-domain antibody specifically binds to CD20 on the surface of Raji cells. The anti-canine CD20 single-domain antibody was subsequently ligated to the canine Fc fragment to construct a camel-canine chimeric antibody. The camel-canine chimeric antibody was expressed in CHO-S cells, and ELISA revealed that the chimeric antibody can specifically bind to the canine CD20 extracellular region polypeptide and the cell surface CD20 in Raji cells. In vitro killing assays revealed that the camel-canine chimeric antibody effectively mediated the targeting of canine peripheral blood mononuclear cells to Raji cells. These results indicate that the camel-canine chimeric antibody has strong anti-canine B-cell lymphoma activity and provide a basis for further development and clinical application of anti-canine B-cell lymphoma drugs.