The design, construction and function of a new chimeric anti-CD20 antibody

摘要

A novel murine IgM-type anti-human CD20 monoclonal antibody (mAb) 1-28 was prepared in our Lab, which can induce apoptosis and inhibit proliferation of Daudi and Raji cells. However, the efficacy of 1-28 mAb in human cancer therapy is likely to be limited by human anti-mouse antibody responses. A chimeric antibody, C1-28, containing 1-28 mAb variable region genes fused to human constant region genes (gamma 1,kappa) was constructed. However, C1-28 lost the antigen-binding activity. Here, using sequence similarity and known 3D structure of antibody variable regions as template, the spatial conformations of 1-28 variable regions (i.e. VH and VL) were analyzed with computer-guided homology modeling methods. According to the surface electrostatic distribution and interaction free energy analysis, the relationship between structure and stability of 1-28 variable regions was studied theoretically and a new chimeric anti-CD20 antibody scFv-lg named 5S was designed. Expression level of 5S in the culture supernatant was determined to be around 50 μg/mL using sandwich ELISA method with chimeric antibody Rituxan as reference. 5S retained its murine counterpart's binding activity by fluorescence-activated cell-sorting analysis. Furthermore, it could kill CD20 positive Daudi and Raji cells by complement-dependent cytotoxicity. For binding affinity often decreased even lost when lgM antibody was constructed into chimeric IgG 1 form, our success give a hint about how to construct a lgG l-type chimeric antibody from lgM-type murine antibody to preserve its binding activity.