The design, construction and function of a new chimeric anti-CD20 antibody.
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 1-28mAb in human cancer therapy is likely to be limited by human anti-mouse antibody responses. A chimeric antibody, C1-28, containing 1-28mAb 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. V(H) and V(L)) 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-Ig named 5S was designed. Expression level of 5S in the culture supernatant was determined to be around 50mug/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 IgM antibody was constructed into chimeric IgG1 form, our success give a hint about how to construct a IgG1-type chimeric antibody from IgM-type murine antibody to preserve its binding activity.
Wang
Yugang
Y
Institute of Basic Medical Sciences, Beijing, PR China.
Feng
Jiannan
J
Huang
Ying
Y
Gu
Xin
X
Sun
Yingxun
Y
Li
Yan
Y
Shen
Beifen
B
D000906:Antibodies / Q000737:chemistry*; D018951:Antigens, CD20 / Q000276:immunology*; D002460:Cell Line; D015195:Drug Design; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007668:Kidney; D008958:Models, Molecular; D050506:Mutant Chimeric Proteins / Q000737:chemistry; D014162:Transfection
D000906:Antibodies; D018951:Antigens, CD20; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D050506:Mutant Chimeric Proteins